Synthesis of prenyloxy coumarin analogues and evaluation of their antioxidant, lipoxygenase (LOX) inhibitory and cytotoxic activity

Article abstract of DOI:10.1007/s00044-017-1800-6

Umbelliferone, 4-methyl-umbelliferone and their farnesyloxy and geranyloxy analogues were synthesized and evaluated for their antioxidant and soybean lipoxygenase inhibitory activity as well as their cytotoxicity against human neuroblastoma cell line SK-N-SH and human hepatoma cell line HepG2. Auraptene (3), followed by 4-methyl-auraptene (4) exhibit modest cytotoxity against the HepG2 cell line. The novel coumarin 6 combines a satisfactory lipoxygenase inhibitory activity with potent cytotoxicity against SK-N-SH cells, but not against HepG2 cells, thus it could be considered as a lead compound for the development of novel therapeutic agents against neuroblastoma.

Full text of DOI:10.1007/s00044-017-1800-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-1800-6
ORIGINAL RESEARCH
Synthesis of prenyloxy coumarin analogues and evaluation of their
antioxidant, lipoxygenase (LOX) inhibitory and cytotoxic activity
Eleni Kavetsou1 Leonidas Gkionis1,2 Georgia Galani1 Christina Gkolfinopoulou2
●
●
●
●
Letta Argyri2 Eleni Pontiki3 Angeliki Chroni2 Dimitra Hadjipavlou-Litina3
●
●
●
●
1
Anastasia Detsi
Received: 28 July 2016 / Accepted: 7 February 2017
© Springer Science+Business Media New York 2017
Abstract Umbelliferone, 4-methyl-umbelliferone and their
farnesyloxy and geranyloxy analogues were synthesized
and evaluated for their antioxidant and soybean lipox-
ygenase inhibitory activity as well as their cytotoxicity
against human neuroblastoma cell line SK-N-SH and
human hepatoma cell line HepG2. Auraptene (3), followed
by 4-methyl-auraptene (4) exhibit modest cytotoxity against
the HepG2 cell line. The novel coumarin 6 combines a
satisfactory lipoxygenase inhibitory activity with potent
cytotoxicity against SK-N-SH cells, but not against HepG2
cells, thus it could be considered as a lead compound for
the development of novel therapeutic agents against
neuroblastoma.
Introduction
Coumarins are heterocyclic organic compounds widely
distributed in the plant kingdom. They exhibit important
biological properties including antioxidant, anticancer,
vasorelaxant, antiviral and anti-inflammatory activities
(Roussaki et al. 2010, 2014).
Umbelliferone (1) (Fig. 1) is a natural coumarin
encountered in numerous plants (Vialart et al. 2012) which
presents remarkable antiparasitic, anticancer and antioxidant
activities. (Chiang et al. 2010; Venugopala et al. 2013;
Ramalingam and Vaiyapuri 2013; Singh et al. 2010).
4-Methyl-umbelliferone (2), is a 7-hydroxy-coumarin
obtained from umbelliferous plants (Apiaceae), including
anise, cumin, parsley, and dill (Kultti et al. 2009; García-
Vilas et al. 2013). 4-Methyl-umbelliferone exhibits a variety
of activities including antioxidant, anticancer (Yates et al.
2015) and antibacterial (Fang et al. 2014). Additionally,
4-methyl-umbelliferone (2) behaves as an antiangiogenic
compound (García-Vilas et al. 2013).
●
●
●
Keywords Coumarin Umbelliprenin Auraptene
●
Lipoxygenase Cytotoxicity
Oxyprenylated products (isopentenyloxy-, geranyloxy-
and the less encountered farnesyloxy-compounds and their
biosynthetic derivatives) represent a family of secondary
metabolites that have been considered for years just as
biosynthetic intermediates of C-prenylated derivatives.
Only in the last decade these natural products have been
recognized as interesting and valuable biologically active
phytochemicals (Alhassan et al. 2014).
Chemical modification such as prenylation, enhances the
structural variety of coumarins and as a result these natural
products represent nowadays a new frontier for the devel-
opment of novel drugs (Roussaki et al. 2014). Pre-
nyloxycoumarins are secondary metabolites commonly
present in plants belonging to the families of Rutaceae and
Umbelliferae. Several of these coumarins were shown to
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-017-1800-6) contains supplementary material,
which is available to authorized users.
* Anastasia Detsi
adetsi@chemeng.ntua.gr
1
Laboratory of Organic Chemistry, School of Chemical
Engineering, National Technical University of Athens, Zografou
Campus, Athens 15780, Greece
2
Institute of Biosciences and Applications, National Centre for
Scientific Research “Demokritos”, Agia Paraskevi, Athens 15310,
Greece
3
Laboratory of Pharmaceutical Chemistry, School of Pharmacy,
Faculty of Health Sciences, Aristotle University of Thessaloniki,
Thessaloniki 54124, Greece

Med Chem Res
Fig. 1 Chemical structures of
umbelliferone (1), 4-methyl-
umbelliferone (hymechromone)
(2), Auraptene (3),
Umbelliprenin (5)
possess valuable pharmacological properties. Among them,
auraptene (7-geranyloxycoumarin, 3) which was first iso-
lated from the peel of citrus fruit (Citrus natsudaidai
Hayata) (Kariyone and Matsuno 1953) has been reported to
have chemopreventive effects on chemically induced car-
cinogenesis (Saldanha et al. 1990). Auraptene (3) is a pro-
mising chemopreventive agent against skin (Murakami
et al. 1997), tongue, esophagus, and colon carcinogenesis in
rodents (Soltani et al. 2010) and can also modulate fat
metabolism (Takahashi et al. 2008). In addition, auraptene
inhibits the production of tumor necrosis factor alpha (TNF-
α) in LPS-stimulated RAW264.7 macrophages (Banbury
et al. 2015). Recently, auraptene was tested as β-lactamase
inhibitor, which is the main cause of resistance against β-
lactam antibiotics, and displayed the most potent inhibitory
activity toward class A β-lactamase among the other tested
compounds (Safdari et al. 2014).
Another natural oxyprenylated coumarin is umbellipre-
nin (7-farnesyloxy-coumarin, 5). Umbelliprenin (5) syn-
thesized by various Ferula plant species (Iranshahi et al.
2009) has been studied for its anticancer activity in different
cancer cell lines (Barthomeuf et al. 2008; Khaghanzadeh
et al. 2012; Valiahdi et al. 2013; Jun et al. 2014; Zhang et al.
2015). Umbelliprenin (5) has also been shown to exhibit an
inhibitory effect on the activity of matrix metalloprotei-
nases, which play critical roles in cancer metastatic cascade,
such as migration, angiogenesis, and invasiveness (Shah-
verdi et al. 2006). In addition, various in vitro and in vivo
models have provided evidence of the antioxidant, anti-
inflammatory (Zamani Taghizadeh Rabe et al. 2016; Iran-
shahi et al. 2009) and cancer chemopreventive properties of
umbelliprenin (Iranshahi et al. 2008). The structures of
coumarins 1, 2, 3, 5 are presented in Fig. 1.
of LT’s (Detsi et al. 2009). Thus, inhibitors of LOX have
attracted attention initially as potential agents for the treat-
ment of inflammatory and allergic diseases, but their ther-
apeutic potential has now been expanded to certain types of
cancer, cardiovascular diseases, acute and chronic inflam-
mation, asthma and rhinitis, arthritis, psoriasis, and heredi-
tary ichthyosis (Haeggstrom and Funk 2011). The majority
of LOX inhibitors are antioxidants or free radical sca-
vengers, since lipoxygenation occurs via a carbon centered
radical, and these compounds can inhibit the formation of
the radical or trap it once formed (Iranshahi et al. 2009;
Kallitsakis et al. 2014; Pontiki and Hadjipavlou-Litina
2007).
As a continuation of our studies concerning the biolo-
gical activity of coumarin analogues (Roussaki et al. 2010,
2014) we present here the synthesis and structural char-
acterization of a series of natural and non-natural coumar-
ins, and the evaluation of their in vitro antioxidant, soybean
LOX inhibitory activities and cytotoxic activity against
human neuroblastoma cell line SK-N-SH and human
hepatoma cell line HepG2.
Results and discussion
Chemistry
In order to efficiently synthesize the desired prenyloxy-
coumarins 3–6, umbelliferone (1, commercially available)
and 4-methyl-7-hydroxy-coumarin (2, synthesized via
Pechmann condensation between resorcinol and ethyl
acetoacetate in the presence of concentrated H2SO4) (Kis-
khan and Yagci 2007) were used as starting materials
(Scheme 1). Alkylation of these coumarins with geranyl and
farnesyl bromide in a basic environment, provided com-
pounds 3–6 in good yields and high purity (Askari et al.
2009). The structures of the synthesized compounds were
identified using NMR spectroscopy.
The study of compounds that encompass two or more
pharmacophores on the same structural framework, is a
well-established approach in medicinal chemistry. Phenolic
acids, such as cinnamic, ferulic, and caffeic acid, account
for an important part of the antioxidant activity displayed by
Lipoxygenases (LOX) are iron-containing enzymes
widely distributed in plants and animals. They catalyze the
oxidation of polyunsaturated fatty acids such as linoleic acid
(in plants) and arachidonic acid (in mammals) at specific
positions to hydroperoxides. In humans LOX plays a key
role in the biosynthesis of leukotrienes (LT’s), the proin-
flammatory mediators mainly released from myeloid cells.
Arachidonic acid is converted to LTA 4 by the enzyme 5-
lipoxygenase. LTA 4 is an unstable intermediate which after
a process of continuing conversion results in the formation

Med Chem Res
Scheme 1 Synthesis of
prenyloxy-coumarins 3–6
Scheme 2 Synthesis of
coumarin 7
fruits, vegetables and several beverages such as juices, wine
and beer together with flavonoid polyphenol phytochem-
icals (Piazzon et al. 2012). Investigation of the potential of
phenolic acids as phytoprotectants revealed that they pos-
sess important anticancer (Gomes et al. 2003; Rocha et al.
2012), cardioprotective (Spilioti et al. 2014) and anti-
inflammatory activity (Leal et al. 2011). Moreover, several
compounds bearing the cinnamate functionality have been
recently shown to potently inhibit soybean LOX activity
(Pontiki et al. 2014; Peperidou et al. 2014). Thus, we
decided to synthesize the ester of cinnamic acid with 4-
methyl-7-hydroxy-coumarin (2) in order to investigate the
impact of this structural feature on the biological activity of
coumarins. The synthesis was straightforward, and involved
the reaction between cinnamoyl-chloride and coumarin 2, in
the presence of triethylamine using tetrahydrofuran (THF)
as a solvent (Scheme 2). The desired ester 7 was obtained as
a white solid without the need of further purification as
shown by NMR spectroscopy.
the Fe3+/ascorbic acid system, expressed as percent inhi-
bition of formaldehyde production, was used for the eva-
luation of their hydroxyl radical scavenging activity.
Finally, the ability of the synthesized coumarins to inhibit
lipid peroxidation (LP) induced by the thermal free radical
producer 2,2’-azobis(2-amidinopropane) dihydrochloride
(AAPH) was evaluated whereas generation of the ABTS•+
(2,2’-azinobis-(3-ethyl-benzothiazoline-6-sulfonic
acid))
radical cation, formed the basis for a further determination
of the antioxidant activity of our compounds (Pontiki et al.
2009, 2011; Roussaki et al. 2010, 2014). The results are
presented in Table 1.
The tested coumarin analogues 1–7 do not possess
satisfactory DPPH radical scavenging ability. This obser-
vation could be attributed firstly to the absence of phenolic
hydroxyl groups on the tested compounds and secondly to
the presence of bulky substituents such as the geranyl and
farnesyl moieties which could prevent effective interaction
of the compounds with the DPPH radical due to steric
hindrance.
Antioxidant activity evaluation
The measured inhibition of LP showed that six out of the
seven tested analogues exhibited significant LP inhibition,
higher than the reference compound trolox. The most effi-
cient LP inhibitor is 4-methyl-umbelliferone (2) (93%),
followed by umbelliferone (1) and umbelliprenin (5). The
presence of a 4-methyl substituent on umbelliprenin, leads
to a less active compound (6) (47%). Auraptene (3) displays
higher activity than its 4-methyl analogue (4). In addition,
auraptene (3) and 4-methyl-auraptene (4) exhibit lower
Taking the multifactorial character of oxidative stress into
account, we decided to evaluate the in vitro antioxidant
activity of the synthesized molecules using four different
antioxidant assays. Therefore, the radical scavenging ability
of the compounds was tested against the 1,1-diphenyl-2-
picryl-hydrazyl (DPPH) stable free radical and the compe-
tition of the compounds with DMSO for HO•, generated by

Med Chem Res
Table 1 Interaction % with DPPH, % inhibition of lipid peroxidation (LP) induced by AAPH, % HO● radical scavenging ability, ABTS●+
decolorization (ABTS●+ %) and inhibition of soybean lipoxygenase (LOX) % at 100 μM or IC50) for compounds 1–7
Compound
clog P (Biobyte Corp.
C-QSAR Database)
DPPH scavenging
ability (%)
(100 μM)
% Inhibition of LP
induced by AAPH
(100 μM)
HO● (%)
(100 μM)
ABTS●+ (%)
(100 μM)
Inhibition of
soybean
lipoxygenase
20 min
60 min
%
ΙC50
(μΜ)
(100 μM)
1
1.58
2.12
5.48
5.98
7.51
8.01
4.29
1
No
26
21
45
31
22
84
–
2
No
6
92
93
52
20
92
47
68
–
n.t.
n.t.
100
49
n.t.
n.t.
no
100
No
44
78
4
41
2
3
4
No
47
4
79
65
5
n.t.
100
83
n.t.
89
6
77
50
86
76
100
4.5
7
2
75
NDGA
Trolox
Ascorbic acid
83
63
73
–
96
n.t. not tested, NDGA Nordihydroguaiaretic acid
inhibition of LP than the corresponding 7-hydroxy analo-
gues 1 and 2, indicating that the presence of the geranyloxy-
moiety does not favor this activity. Esterification of the free
hydroxyl-group of 4-methyl-umbelliferone with cinnamic
acid leads to compound 7 with satisfactory LP inhibitory
activity (68%), albeit lower than the starting 4-methyl-
umbelliferone (2).
The most active LOX inhibitor among the tested cou-
marin derivatives is umbelliferone (1) followed by 4-
methyl-auraptene (4) (41 μΜ, 65 μM, respectively) and 4-
methyl-umbelliprenin (6) (IC50 76 μΜ).
It is noteworthy that insertion of a methyl group at
position 4 of the umbelliferone structure (compound 2)
leads to complete loss of activity whereas in the case of
auraptene (3), the presence of methyl group enhanced the
LOX inhibitory activity (compound 4). Respectively, the
novel compound 4-methyl-umbelliprenin (6) is a better
inhibitor of LOX in comparison with umbelliprenin (5).
Cinnamic ester (7) displays lower activity as inhibitor of
LOX (IC50 100 μΜ). Although lipophilicity is a major
physicochemical property influencing activity, the presented
results do not support this idea. On the contrary steric
effects seem to be more significant.
Hydroxyl radicals are among the most reactive oxygen
species and are considered to be in part responsible for
tissue damage occurring in inflammation (Pontiki and
Hadjipavlou-Litina 2007). Auraptene (3) and 4-methyl-
umbelliprenin (6) are very effective HO● scavengers
(100%) whereas ester 7 also shows satisfactory activity
against this free radical.
Coumarin 6 is the most potent ABTS●+ radical cation
scavenger among the compounds tested in this work, fol-
lowed by 4-methyl-auraptene (4) and ester 7.
Anti-inflammatory activity evaluation
Glutathione conjugation
Furthermore, the ability of the compounds to inhibit soy-
bean LOX, using the UV absorbance based enzyme assay,
was determined as an indication of potential anti-
inflammatory activity (Kontogiorgis and Hadjipavlou-
Litina 2003). The results are presented in Table 1.
Although the results of this assay cannot be extrapolated to
the inhibition of mammalian 5-LOX, it has been shown that
inhibition of plant LOX activity by nonsteroidal anti-
inflammatory agents is qualitatively similar to the inhibition
they cause to the rat mast cell LOX. Therefore, the soybean
inhibition assay can be used as a simple qualitative assay for
such activity.
Glutathione (GSH) conjugation is an important pathway by
which reactive electrophilic compounds are detoxified. It
protects vital cellular constituents against chemical reactive
species by virtue of its nucleophilic sulfhydryl group and
constitutes an in vivo antioxidant protective mechanism.
The nucleophilic addition of GSH to electron-deficient
carbon double bonds occurs mainly in compounds with α,β-
unsaturated double bonds. In most instances the double
bond is rendered electron deficient by resonance or con-
jugation with a carbonyl group. The coumarins synthesized
in this study were tested for their ability to react with GSH
in vitro and the results are presented in Table 2.

Med Chem Res
Table 2 Study of the conjugation of coumarins 3, 4, 6 and 7 with
GSH
Conclusions
Compound
λmax
εmax
In conclusion, umbelliferone, hymechromone and their
farnesyloxy and geranyloxy analogues as well as the ester
of cinnamic acid with 7-hydroxy-4-methyl-coumarin, were
synthesized and tested for their antioxidant and cytotoxic
activity. The results of this study indicate that umbelliferone
(1), 4-methyl-auraptene (4) and the new analogue 4-methyl-
umbelliprenin (6) efficiently inhibit soybean LOX while
umbelliferone (1) is also a potent LP inhibitor. Auraptene
(3) exhibits the most potent cytotoxic activity, followed by
4-methyl-auraptene (4), umbelliprenin (5) and 4-methyl-
umbelliprenin (6) against the SK-N-SH cell line thus they
could be considered as drug candidates for neuroblastoma
therapy after additional evaluation. Auraptene (3), followed
by 4-methyl-auraptene (4) exhibit modest cytotoxity against
the HepG2 cell line, whereas all other coumarin analogs are
not cytotoxic against HepG2. Overall, as it has been shown
that inhibition of the leukotriene pathway leads to apoptosis
of neuroblastoma cells in vitro (Sveinbjornsson et al. 2008)
the novel coumarin 6, which combines a satisfactory LOX
inhibitory activity with potent cytotoxicity against the SK-
N-SH line and a very significant antioxidant profile, should
be further explored as a lead compound for the development
of novel therapeutic agents against neuroblastoma.
3
325
325
325
322
322
322
322
322
322
329
329
329
136
148
143
516
738
715
261
384
359
318
543
368
3 + 2GSH
3 + 10GSH
4
4 + 2GSH
4 + 10GSH
6
6 + 2GSH
6 + 10GSH
7
7 + 2GSH
7 + 10GSH
GSH Glutathione
No conjugation was observed between the tested com-
pounds and GSH. This was supported by the determined
εmax values at λmax.. It is most likely that stereochemistry
affects the conjugation process.
Cytotoxic activity evaluation
Coumarins have been reported to exhibit toxicity against
different types of cancer cells. The investigation of potential
cytotoxicity by our compounds was measured by 3-(4,5-
Experimental
Chemicals and instruments
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
(MTT) assay using the human neuroblastoma cell line SK-
N-SH and the human hepatoma cell line HepG2 (Berridge
and Tan 1993) The results are presented in Figs. 2 and 3.
The natural coumarin auraptene (3) is the most potent
cytotoxic coumarin among the studied compounds in both
cell lines. The farnesyloxy coumarin umbelliprenin (5)
shows cytotoxicity only on the SK-N-SH cell line whereas
it is not cytotoxic on HepG2 cells. 4-methyl-auraptene (4)
possesses decreased cytotoxic activity on both cell lines as
compared to auraptene (3). In the case of 4-methyl-
umbelliprenin (6) the cytotoxicity on SK-N-SH cells was
similar to that of umbelliprenin (5), however the compound
shows a moderate cytotoxicity on HepG2 cells.
The chemicals used for synthesis and analysis were pur-
chased from Sigma-Aldrich or Alfa Aesar (7-hydro-
xycoumarin, 98%) and used without further purification.
NMR spectra were recorded on a Varian 300 MHz spec-
trometer at the National Hellenic Research Foundation. The
HR-MS spectrum was obtained using a UHPLC-MSn
Orbitrap Velos-Thermo mass spectrometer. Melting points
were determined on a Gallenkamp MFB-595 melting point
apparatus and are uncorrected.
Synthesis of 4-methyl-7-hydroxy coumarin (4 methyl
umbelliferone) (2)
Umbelliferone (1) and hymechromone (2) affect cell
viability only at high concentrations thus, as far as cyto-
toxicity against SK-N-SH and HepG2 cells is concerned,
specific alkylation of the 7-OH group significantly enhances
cytotoxic activity. On the other hand, esterification of the 7-
OH of hymechromone with cinnamic acid leads to a com-
pound which is not cytotoxic on either cell line (coumarin
analogue 7).
The desired compound was prepared following the method
of Kiskhan and Yagci (Kiskhan and Yagci 2007) Yield:
65%; m.p.:186–187 °C; H NMR (300 ΜΗz, DMSO-d6): δ
(ppm) 10.47 (s, 1H, 7-OH), 7.56 (d, J = 8.7 Hz, 1H, H-5),
6.77 (dd, J6,8 = 1.8 Hz, J 6,5 = 8.4 Hz, 1H, H-6), 6.68 (d, J
8,6 = 1.8 Hz, 1H, H-8), 6.10 (s, 1H, H-3), 2.36 (s, 3H, 4-
CH3).
1

Med Chem Res
Fig. 2 Effect of coumarin analogues on human neuroblastoma cell
SK-N-SH viability. Shown is the survival of SK-N-SH cells incubated
with coumarin analogs at various concentrations for 48 h, as deter-
mined by a MTT assay. 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 < 0.05 vs. control; **p < 0.005 vs. control; ***p < 0.0005 vs.
control

Med Chem Res
Fig. 3 Effect of coumarin analogues on human hepatoma cell line
HepG2 viability. Shown is the survival of HepG2 cells incubated with
coumarin analogs at various concentrations for 24 h, as determined by
a MTT assay. Cell viability is expressed as percent relative to the
viability of control untreated cells (0 μM, incubation with the com-
pounds dilution medium) set to 100%. Data are the means SD *p <
0.05 vs. control; **p < 0.005 vs. control; ***p < 0.0005 vs. control
General procedure for the synthesis of prenyloxy-
coumarins 3–6
the completion of the reaction, potassium carbonate was
filtrated, the precipitate was washed with acetone and the
solvent was removed under reduced pressure. The products
were purified by recrystallization.
7-geranyloxy-coumarin (auraptene) (3) was prepared
following the general procedure, starting from 0.0015 mol
A mixture of the appropriate 7-hydroxy-coumarin and far-
nesyl or geranyl-bromide in acetone in the presence of
potassium carbonate (K2CO3) was refluxed overnight. After

Med Chem Res
(0.25 g) 7-hydroxy-coumarin (1), 0.002 mol (0.4 mL) ger-
anyl bromide and 0.0015 mol (0.21g) K2CO3 in 21.6 mL
acetone. The white solid product was collected after
recrystallization from methanol and hexane. Yield: 52%, m.
p.: 66–67 °C (lit. m.p. 62.7–63.4 °C) (Askari et al. 2009).
1H NMR (300 MHz, CDCl3): δ (ppm) 7.63 (d, J = 9.3 Hz,
1H, H-4), 7.36 (d, J = 8.4, 1H, H-5), 6.86–6.82 (m, 2H, H-6
& H-8), 6.24 (d, J = 9.3 Hz, 1H, H-3), 5.47 (t, J = 6 Hz, 1H,
H-2′), 5.09 (t, J = 5.7 Hz, 1H, H-6′), 4.62 (d, J = 6.6 Hz,
2H, Η-1′), 2.15-2.11 (m, 4H, H-4′ & H-5′), 1.79 (s, 3H, 3′-
CH3), 1.69 (s, 3H, 8′-CH3), 1.63 (s, 3H, 7′-CH3).
155.14 (C-8a), 153.85 (C-4), 141.46 (C-4), 135.14 (C-3′),
131.06 (C-7′), 126.79 (C-11′), 124.52 (C-5), 123.89 (C-6′ &
C-10′), 119.52 (C-2′), 113.45 (C-4a), 113.05 (C-6), 111.48
(C-3), 101.82 (C-8), 65.55 (C-1′), 39.67 (C-8′), 39.30 (C-
4′), 26.58 (C-9′), 25.99 (C-5′), 25.92 (12′-CH3), 18.56 (4-
CH3), 17.96 (3′-CH3), 16.85 (8”-CH3), 16.27 (11′-CH3).
HRMS calcd for C25H32O3 Na: m/z: 403.2244, found:
403.2245.
4-methyl-2-oxo-2H-chromen-7-yl cinnamate (7)
4-methyl-7-geranyloxy-coumarin (4-methyl-auraptene)
(4): Prepared following the general procedure, starting from
0.0014 mol (0.25 g) 4-methyl-7-hydroxy- coumarin (2),
0.0015 mol (0.3 mL) geranyl bromide and 0.0014 mol
(0.19 g) of K2CO3 in 19.9 mL acetone. The brown gummy
solid product was collected after recrystallization from
0.00142 mol (0.25 g) 4-methyl-7-hydroxy-coumarin (2),
0.00213 mol (0.356 g) cinnamoyl chloride, and 5.5 mL
triethylamine (Εt3Ν) were added in 7.1 mL dry THF and
stirred for 24 h. After the reaction is adding a suitable
amount of water, followed by extracting the mixture with
dichloromethane (CH2Cl2) The organic phase was dried
over anhydrous sodium sulfate (Na2SO4), filtered and eva-
porated to give the final white solid product in high purity.
Yield: 77%, m.p.: 145–146 °C (lit. m.p.: 149–151 °C)
(Nakayama and Kanaoka 1973). 1H NMR (300 MHz,
CDCl3): δ (ppm) 7.91 (d, J = 15.9 Hz, 1H, H-b), 7.65-7.59
(m, 3H, H-6′ & H-2′), 7.45-7.43 (m, 3H, H-3′ & H-5′ & H-
4′), 7.21 (d, J = 2.1 Hz, 1H, H-8), 7.17 (dd, J = 2.1 Hz, J =
8.4 Hz, 1H, H-6), 6.65 (d, J = 15.9 Hz, 1H, H-a), 6.29 (s,
1H, H-3), 2.47 (s, 3H, 4-CH3).
1
methanol (3 mL) and hexane (1 mL). Yield: 68%, H NMR
(300 MHz,DMSO-d6): δ (ppm) 7.64 (d, J = 8.4 Hz, 1H, Η-
5), 6.94–6.91 (m, 2H, H-6 & H-8), 6.18 (s, 1H, H-3), 5.42
(t, J = 6 Hz, 1H, H-2’), 5.03 (br, 1H, H-6′), 4.65 (d, J = 6.6
Hz, 2H, Η-1′), 2.39 (s, 3H, 4-CH3), 2.08–2.06 (m, 4H, H-4′
& H-5′), 1.73 (s, 3H, 3′-CH3), 1.61 (s, 3H, 8′-CH3), 1.56 (s,
3H, 7′-CH3).
7-farnesyloxy- coumarin (Umbelliprenin) (5): Prepared
following the general procedure, starting from 0.0015 mol
(0.25 g) 7-hydroxy- coumarin (1), 0.00185 mol (0.5 mL)
farnesyl bromide and 0.0015 mol (0.21g) K2CO3 in 21.6
mL acetone. The yellow solid product was collected after
recrystallization from methanol (3 mL) and hexane (1 mL).
Yield: 30%, m.p.: 61–63 °C (lit. m.p.: 57.5–59.1 °C)
Determination of the reducing activity of the stable
radical DPPH
To an ethanolic solution of DPPH (100 µM) in absolute
ethanol an equal volume of the compounds dissolved in
DMSO was added (100 µM). The mixture was shaken
vigorously, in some cases with the help of ultrasound, and
then allowed to stand for 20 or 60 min; absorbance at 517
nm was determined spectrophotometrically and the per-
centage of activity was calculated. All tests were undertaken
on three replicates and the results were averaged and
compared with the appropriate standard NDGA (Table 1)
(Hadjipavlou et al. 2009).
1
(Askari et al. 2009). H NMR (300 MHz, CDCl3): δ (ppm)
7.62 (d, J = 9.3 Hz, 1H, H-4), 7.35 (d, J = 8.1 Hz, 1H, H-5),
6.86-6.82 (m, 2H, H-6 & H-8), 6.24 (d, J = 9.6 Hz, 1H, H-
3), 5.48 (t, J = 6.6 Hz , 1H, H-2′), 4.62 (d, J = 6.6 Hz , 1H,
H-1′), 2.18 -2.01 (m, 8H, H-4′ & H-5′ & H-8′ & H-9′), 1.79
(s, 3H, 3′-CH3), 1.70 (s, 3H, 8′-CH3), 1.63 (s, 6H, 11′-CH3
& 12′-CH3).
4-methyl-7-farnesyloxy-coumarin (4-methyl-Umbelli-
prenin) (6): Prepared following the general procedure,
starting from 0.00142 mol (0.25 g) 4-methyl-7-hydroxy-
coumarin (2), 0.0017(0.46 mL) farnesyl bromide and
0.00142 mol (0.196 g) K2CO3 in 19.9 mL acetone. The
yellow gummy solid product was collected after recrys-
tallization from methanol and hexane. Yield: 50%.1H NMR
(300 MHz, CDCl3): δ (ppm) 7.48 (d, J = 8.7 Hz, 1H, H-5),
6.86 (d, J6,8 = 1.8 Hz, J6,5 = 8.7 Hz, 1H, H-6), 6.82
(d, J8,6 = 1.8 Hz, 1H, H-8), 6.13 (s, 1H, H-3) , 5.47 (t, J =
6.9 Hz, 1H, H-2′), 5.10- 5.07 (m, 1H, H-6′), 4.60 (d, J = 6.6
Hz, 1H, H-1′), 2.39 (s, 3H, 4-CH3), 2.12–1.95 (m, 8H, H-4′
& H-5′ & H-8′ & H-9′), 1.76 (s, 3H, 3′-CH3), 1.67 (s, 3H,
8′-CH3), 1.59 (s, 6H, 11′-CH3 & 12′-CH3); 13C NMR (75
MHz, DMSO-d6): δ (ppm) 161.95 (C-2), 160.59 (C-7),
Inhibition of linoleic acid lipid peroxidation
Production of conjugated diene hydroperoxide by oxidation
of linoleic acid in an aqueous dispersion is monitored at
234 nm. AAPH is used as a free radical initiator. Ten
microliters of the 16 mM linoleic acid sodium salt solution
was added to the UV cuvette containing 0.93 mL of 0.05 M
phosphate buffer, pH 7.4 prethermostated at 37 °C. The
oxidation reaction was initiated at 37 °C under air by the
addition of 50 µL of 40 mM AAPH solution. Oxidation was
carried out in the presence of coumarins (10 µL, from a
stock solution of 10 mM in DMSO) in the assay. Lipid

Med Chem Res
oxidation was measured in the presence of the same level of
DMSO. The rate of oxidation at 37 °C was monitored by
recording the increase in absorption at 234 nm caused by
conjugated diene hydroperoxides and compared
with the appropriate standard trolox (Hadjipavlou et al.
2009).
Glutathione conjugation assay
Stock solutions of the compounds were prepared in water
using phosphates buffer solution (PBS) pH 7.4 and in order
to achieve dissolution the solvent contained approximately
10% DMSO. The concentrations of the solutions were
chosen so that the absorption maxima were between 0.5 and
1. The test compounds are incubated for 24 h at 37 °C and
their UV spectra were recorded. All determinations were
carried out in duplicate. The error limits of the ε values were
approximately 2%. The experiment was repeated in the
presence of GSH using thiol/test compound, 2/1 and 10/1
and incubation at 37 °C for 24 h and their UV spectra were
recorded. (Pontiki and Hadjipavlou-Litina 2007).
Competition of the tested compounds with DMSO for
hydroxyl radicals
The hydroxyl radicals generated by the Fe3+/ascorbic acid
system, were detected by the determination of for-
maldehyde produced from the oxidation of DMSO. The
reaction mixture contained EDTA (0.1 mM), Fe3+ (167
mM), DMSO (33 mM) in phosphate buffer (50 mM, pH
7.4), the tested compounds and ascorbic acid (10 mM).
After 30 min of incubation at 37 °C the reaction was stop-
ped with CCl3COOH (17% w/v) and the % competition of
the tested compounds with DMSO for hydroxyl radicals
was determined (Table 1) (Pontiki and Hadjipavlou-Litina
2006).
Cell viability assay
Cell viability was measured by 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma) assay
(Berridge and Tan 1993). Human neuroblastoma SK-N-SH
cells (ATCC) were cultured in minimal essential medium
(MEM) Earle’s supplemented with 2 mM L-glutamine, 0.1
mM nonessential amino acids, 1 mM sodium pyruvate, 1.5
g/liter sodium bicarbonate, 10% FBS and antibiotics.
Human hepatoma HepG2 cells (ATCC) were cultured in
Dulbecco’s Modified Eagle’s medium (DMEM) (high glu-
cose), 10% FBS and antibiotics. The cells were seeded in 96
well plates at a density of 2 × 104 cells/well in complete cell
culture medium. The coumarin compounds were dissolved
in DMSO and then diluted at the appropriate concentration
in the culture medium. The final DMSO concentration in
each well was 0.5% (v/v). Twenty four hours after seeding,
the media were changed into serum-free medium containing
the compounds, at increasing concentrations of 5, 10, 50,
100, 250, and 500 μΜ, and the cells were incubated for 24 h
(HepG2) or 48 h (SK-N-SH) at 37 °C. At the end of the
incubation, the cells were further incubated in serum-free
medium containing 0.65 mg/ml MTT for 3 h at 37 °C. Then,
the medium was removed, DMSO was added in each well
to dissolve the dark blue formazan crystals formed by
metabolically active cells containing functional mitochon-
dria and absorbance was read at 570 nm. At least two
independent experiments were done in triplicate for each
compound and the IC50 values were calculated using the
GraphPad Prism software.
ABTS•+—decolorization assay for antioxidant activity
ABTS was dissolved in water to a 7 mM concentration.
ABTS radical cation (ABTS•+) was produced by reacting
ABTS stock solution with 2.45 mM potassium persulfate
and allowing the mixture to stand in the dark at room
temperature for 12e16 h before use. For the present study,
the ABTS•+ solution was diluted with ethanol to an
absorbance of 0.70 at 734 nm and equilibrated at 30 °C.
Stock solutions of the tested compounds in DMSO were
diluted so that, after introduction of a 10 mL aliquot of each
dilution into the assay, they produced between 20 and 80%
inhibition of the blank absorbance. After addition of 1.0 mL
of diluted ABTS•+ solution (λ = 734 nm) to 10 mL of
antioxidant compounds or Trolox standards (final con-
centration 0–0.1 mM) in ethanol the absorbance reading
was taken at 30 °C exactly 1 min after the initial mixing
(Table 1).
Soybean LOX inhibition study in vitro
The tested compounds dissolved in DMSO were incubated
at room temperature with sodium linoleate (0.1 ml) and
0.2 ml of enzyme solution (1/9 × 10−4 w/v in saline). The
conversion of sodium linoleate to 13-hydroperoxylinoleic
acid at 234 nm was recorded and compared with the
appropriate standard inhibitor NDGA (Hadjipavlou et al.
2009).
Acknowledgements Cytotoxicity assays were performed using
facilities that are associated with the OPENSCREEN-GR National
(Greece) Research Infrastructure. The authors are grateful to Dr. A.
Leo and Biobyte Corp. 201 West 4th Str., Suite 204, Claremont CA
California, 91711, USA for free access to the C-QSAR program.

Med Chem Res
Compliance with ethical standards
coumarin, umbelliprenin, in vivo. Eur J Cancer Prevention
18:412–415
Jun M, Bacay AF, Moyera J, Webbb A, Carrico-Moniz D (2014)
Synthesis and biological evaluation of isoprenylated coumarins as
potential anti-pancreatic cancer agents. Bioorg Med Chem Lett
24:4654–4658
Conflict of interest The authors declare that they have no competing
interest.
Kallitsakis MG, Hadjipavlou-Litina DJ, Peperidou A, Litinas KE
(2014) Synthesis of 4-hydroxy-3-[(E)-2-(6-substituted-9H-purin-
9-yl)vinyl]-coumarins as lipoxygenase inhibitors. Tetrahedron
Lett 55:650–653
Kariyone T, Matsuno T (1953) Studies on the constituents of orange
oil. I. On the structure of auraptene. Pharm Bull 1:119–122
Khaghanzadeh N, Mojtahedi Z, Ramezani M, Erfani N, Ghaderi A
(2012) Umbelliprenin is cytotoxic against QU-DB large cell lung
cancer cell line but anti-proliferative against A549 adenocarci-
noma cells. DARU J Pharm Sci 20:69
References
Alhassan AM, Abdullahi MI, Uba A, Umar A (2014) Review article
prenylation of aromatic secondary metabolites: a new frontier for
development of novel drugs. Trop J Pharm Res 13:307–314
Askari M, Sahebkar A, Iranshahi M (2009) Synthesis and purification
of 7-prenyloxycoumarins and Herniarin as bioactive natural
coumarins. Iranian J Basic Medical Sci 12:63–69
Banbury LK, Shou Q, Renshaw DE, Lambley EH, Griesser HJ, Monb
H, Wohlmuth H (2015) Compounds from Geijera parviflora with
prostaglandin E2 inhibitory activity may explain its traditional
use for pain relief. J Ethnopharmacol 163:251–255
Kiskhan B, Yagci Y (2007) Thermally curable benzoxazine monomer
with
a photodimerizable coumarin group. J Polym Sci
45:1670–1676
Kontogiorgis C, Hadjipavlou-Litina D (2003) Biological evaluation of
several coumarin derivatives designed as possible anti-inflam-
matory/antioxidant agents. J Enzyme Inhib Med Chem 18:63–69
Kultti A, Pasonen-Seppanen S, Jauhiainen M, Rilla KJ, Karna R,
Pyoria E, Tammi RH, Tammi MI (2009) 4-Methylumbelliferone
inhibits hyaluronan synthesis by depletion of cellular UDP-
glucuronic acid and downregulation of hyaluronan synthase 2 and
3. Exp Cell Res 315:1914–1923
Leal LKAM, Pierdon TM, Goes JGS, Fonsκca KS, Canuto KM, Sil-
veira ER, Bezerra AME, Viana GSB (2011) A comparative
chemical and pharmacological study of standardized extracts and
vanillic acid from wild and cultivated Amburana cearensis A.C.
Smith. Phytomedicine 18:230–233
Murakami A, Kuki W, Takahashi Y, Yonei H, Nakamura Y, Ohto Y,
Ohigashi H, Koshimizu K (1997) Auraptene, a citrus coumarin,
inhibits 12-O-tetradecanoylphorbol-13-acetate-induced tumor
promotion in ICR mouse skin, possibly through suppression of
superoxide generation in leukocytes. Japanese J Cancer Res
88:443–452
Nakayama H, Kanaoka Y (1973) Spectrofluorometric titrants for
trypsin and chymotrypsin. Chem Pharm Bull 21:2804–2805
Peperidou A, Kapoukranidou D, Kontogiorgis C, Hadjipavlou-Litina
D (2014) Multitarget molecular hybrids of cinnamic acids.
Molecules 19:20197–20226
Barthomeuf C, Lim S, Iranshahi M, Chollet P (2008) Umbelliprenin
from Ferula szowitsiana inhibits the growth of human M4Beu
metastatic pigmented malignant melanoma cells through cell-
cycle arrest in G1 and induction of caspase-dependent apoptosis.
Phytomedicine 15:103–111
Berridge MV, Tan AS (1993) Characterization of the cellular reduction
of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT): subcellular localization, substrate dependence, and
involvement of mitochondrial electron transport in MTT reduc-
tion. Arch Biochem Biophys 303:474–482
Biobyte Corp., C-QSAR Database, 201 West 4th Str., Suite 204,
Claremont CA, California 91711, USA
Chiang CC, Cheng MJ, Peng CF, Huang HY, Chen IS (2010) A novel
dimeric coumarin analog and antimycobacterial constituents from
Fatoua pilosa. Chem Biodiv 7:1728–1736
Detsi A, Majdalani M, Kontogiorgis CA, Hadjipavlou-Litina D,
Kefalas P (2009) Natural and synthetic 2’-hydroxy-chalcones and
aurones: synthesis, characterization and evaluation of the anti-
oxidant and soybean lipoxygenase inhibitory activity. Bioorg
Med Chem 17:8073–8085
Fang Y, Wang H, Zhu W, Wang L, Liu H, Xu X, Yin W, Sima Y, Xu
S (2014) Antioxidative properties of 4 methylumbelliferone are
related to antibacterial activity in the silkworm (Bombyx mori)
digestive tract. J Comp Physiol B 184:699–708
Piazzon A, Vrhovsek U, Masuero D, Mattivi F, Mandoj F, Nardini M
(2012) Antioxidant activity of phenolic acids and their metabo-
lites: synthesis and antioxidant properties of the sulfate deriva-
tives of ferulic and caffeic acids and of the acyl glucuronide of
ferulic acid. J Agric Food Chem 60:12312–12323
Pontiki E, Hadjipavlou-Litina D (2007) Synthesis and pharmaco-
chemical evaluation of novel aryl-acetic acid inhibitors of
lipoxygenase, antioxidants, and anti-inflammatory agents. Bioorg
Med Chem 15:5819–5827
́
García-Vilas JA, Quesada AR, Medina MA (2013) 4-
Methylumbelliferone inhibits angiogenesis in vitro and in vivo.
J Agric Food Chem 61:4063–4071
Gomes CA, Girao da Cruz T, Andrade JL, Milhazes N, Borges F,
Marques MPM (2003) Anticancer activity of phenolic acids of
natural or synthetic origin: a structure-activity study. J Med Chem
46:5395–5401
Hadjipavlou D, Garnelis T, Athanassopoulos CM, Papaioannou D
(2009) Kukoamine
a analogs with lipoxygenase inhibitory
Pontiki E, Hadjipavlou-Litina
D (2006) Antioxidant and anti-
activity. J Enzyme Inhib Med Chem 24:1188–1193
Haeggstrom JZ, Funk CD (2011) Lipoxygenase and Leukotriene
Pathways: Biochemistry, Biology, and roles in disease. Chem
Rev 111:5866–5898
Iranshahi M, Askari M, Sahebkar A, Hadjipavlou-Litina D (2009)
Evaluation of antioxidant, anti-inflammatory and lipoxygenase
inhibitory activities of the prenylated coumarin umbelliprenin.
DARU 17:99–103
inflammatory activity of aryl-acetic and hydroxamic acids as
novel lipoxygenase inhibitors. Med Chem 2:251–264
Pontiki E, Hadjipavlou-Litina D, Geromichalos G, Papageorgiou A
(2009) Anticancer activity and quantitative-structure activity
relationship (QSAR) studies of a series of antioxidant/anti-
inflammatory aryl-acetic and hydroxamic acids. Chem Biol Drug
Des 74:266–275
Pontiki E, Hadjipavlou-Litina D, Litinas K, Geromichalos G (2014)
Novel cinnamic acid derivatives as antioxidant and anticancer
agents: design, synthesis and modeling studies. Molecules
19:9655–9674
Pontiki E, Hadjipavlou-Litina D, Litinas K, Nicolotti O, Carotti A
(2011) Design, synthesis and pharmacobiological evaluation of
Iranshahi M, Kalategi F, Rezaee R, Shahverdi AR, Ito C, Furukawa H,
Tokuda H, Itoigawa M (2008) Cancer chemopreventive activity
of terpenoid coumarins from Ferula species. Planta Med
74:147–150
Iranshahi M, Sahebkar A, Takasaki M, Konoshima T, Tokuda H
(2009) Cancer chemopreventive activity of the prenylated

Med Chem Res
novel acrylic acid derivatives acting as lipoxygenase and
cyclooxygenase-1 inhibitors with antioxidant and anti-
inflammatory activities. Eur J Med Chem 46:191–200
Ramalingam R, Vaiyapuri M (2013) Effects of umbelliferone on lipid
peroxidation and antioxidant status in diethylnitrosamine-induced
hepatocellular carcinoma. J Acute Med 3:73–82
Sveinbjornsson B, Rasmuson A, Baryawno N, Wan M, Pettersen I,
Ponthan F, Orrego A, Haeggstrom JZ, Johnsen JI, Kogner P
(2008) Expression of enzymes and receptors of the
leukotriene pathway in human neuroblastoma promotes tumor
survival and provides
a target for therapy. FASEB J
22:3525–3536
Rocha LD, Monteiro MC, Teodoro A (2012) Anticancer properties of
hydroxycinnamic acids: a review. J Cancer Clin Oncol 1:109–121
Roussaki M, Kontogiorgis CA, Hadjipavlou-Litina D, Hamilakis S,
Detsi A (2010) A novel synthesis of 3-aryl coumarins and eva-
luation of their antioxidant and lipoxygenase inhibitory activity.
Bioorg Med Chem Lett 20:3889–3892
Roussaki M, Zelianaios K, Kavetsou E, Hamilakis S, Hadjipavlou-
Litina D, Kontogiorgis C, Liargkova T, Detsi A (2014) Structural
modifications of coumarin derivatives: determination of anti-
oxidant and lipoxygenase (LOX) inhibitory activity. Bioorg Med
Chem 22:6586–6594
Safdari H, Neshani A, Sadeghian A, Ebrahimi M, Iranshahi M,
Sadeghian H (2014) Potent and selective inhibitors of class A b-
lactamase:7-prenyloxy coumarins. J Antibiotics 67:373–377
Saldanha LA, Elias G, Gao MNA (1990) Oxygen radical scavenging
activity of phenylbutenones and their correlation with antiin-
flammatory activity. Arzneim.-forsch/Drug Res 40:89–91
Shahverdi AR, Saadat F, Khorramizadeh MR, Iranshahi M, Khosh-
ayand MR (2006) Two matrix metalloproteinases inhibitors from
Ferula persica var. persica. Phytomedicine 13:712–717
Singh R, Singh B, Singh S, Kumar N, Kumar S, Arora S (2010)
Umbelliferone—an antioxidant isolated from Acacia nilotica (L.)
Willd Ex Del Food Chem 120:825–830
Soltani F, Mosaffa F, Iranshahi M, Karimi G, Malekaneh M, Haghighi
F, Behravan J (2010) Auraptene from Ferula szowitsiana protects
human peripheral lymphocytes against oxidative stress. Phy-
totherapy Res 24:85–89
Spilioti E, Jaakkola M, Tolonen T, Lipponen M, Virtanen V, Chinou I,
Kassi E, Karabournioti S, Moutsatsou P (2014) Phenolic acid
composition, antiatherogenic and anticancer potential of honeys
derived from various regions in Greece. PLOS 9:e94860
Takahashi N, Kang M, Kuroyanagi K, Goto T, Hirai S, Ohyama K,
Lee J, Yu R, Yano M, Sasaki T, Murakami S, Kawada T (2008)
Auraptene, a citrus fruit compound, regulates gene expression as
a
PPAR alpha agonist in HepG2 hepatocytes. Biofactors
33:25–32
Valiahdi SM, Iranshahi M, Sahebkar A (2013) Cytotoxic activities of
phytochemicals from Ferula species. DARU J Pharm Sci 21:39
Vialart G, Hehn A, Olry A, Ito K, Krieger C, Larbat R, Paris C,
Shimizu B, Sugimoto Y, Mizutani M, Bourgaud F (2012) A 2-
oxoglutarate-dependent dioxygenase from Ruta graveolens L.
exhibits p-coumaroyl CoA 2’-hydroxylase activity (C2’H): a
missing step in the synthesis of umbelliferone in plants. Plant J
70:460–470
Venugopala KN, Rashmi V, Odhav B (2013) Review on natural
coumarin lead compounds for their pharmacological activity.
BioMed Res Int. Article ID 963248, p 14, http://dx.doi.org/10.
1155/2013/963248
Yates TJ, Lopez LE, Lokeshwar SD, Ortiz N, Kallifatidis G,
Jordan A, Hoye K, Altman N, Lokeshwar VB (2015) Dietary
supplement 4-methylumbelliferone: an effective chemopreventive
and therapeutic agent for prostate cancer. J Natl Cancer Inst 107:
djv085
Zamani Taghizadeh Rabe S, Iranshahi M, Mahmoudi M (2016) In
vitro anti-inflammatory and immunomodulatory properties of
umbelliprenin and methyl galbanate.
J
Immunotoxicol
13:209–216
Zhang L, Si J, Li G, Li X, Zhang L, Li Gao Li, Huo X, Liu D, Suna X,
Cao L (2015) Umbelliprenin and lariciresinol isolated from a
long-term-used herb medicine Ferula sinkiangensis induce
apoptosis and G0/G1 arresting in gastric cancer cells. RSC Adv
5:91006–91017