Ijms 21 04618 v2

International Journal of
Molecular Sciences
Review
An Overview of Coumarin as a Versatile and Readily
Accessible Scaffold with Broad-Ranging
Biological Activities
Francesca Annunziata 1,† , Cecilia Pinna 1,† , Sabrina Dallavalle 2 , Lucia Tamborini 1, * and
Andrea Pinto 2
1 Department of Pharmaceutical Science, University of Milan, via Mangiagalli 25, 20133 Milan, Italy;
francesca.annunziata@unimi.it (F.A.); cecilia.pinna@unimi.it (C.P.)
2 Department of Food, Environmental and Nutritional Sciences, University of Milan, via Celoria 2,
20133 Milan, Italy; sabrina.dallavalle@unimi.it (S.D.); andrea.pinto@unimi.it (A.P.)
* Correspondence: lucia.tamborini@unimi.it; Tel.: +39-0250319367
† These authors contributed equally to this work.

Received: 31 May 2020; Accepted: 28 June 2020; Published: 29 June 2020
Abstract: Privileged structures have been widely used as an effective template for the research and
discovery of high value chemicals. Coumarin is a simple scaffold widespread in Nature and it can be
found in a considerable number of plants as well as in some fungi and bacteria. In the last years, these
natural compounds have been gaining an increasing attention from the scientific community for their
wide range of biological activities, mainly due to their ability to interact with diverse enzymes and
receptors in living organisms. In addition, coumarin nucleus has proved to be easily synthetized and
decorated, giving the possibility of designing new coumarin-based compounds and investigating their
potential in the treatment of various diseases. The versatility of coumarin scaffold finds applications
not only in medicinal chemistry but also in the agrochemical field as well as in the cosmetic and
fragrances industry. This review is intended to be a critical overview on coumarins, comprehensive
of natural sources, metabolites, biological evaluations and synthetic approaches.
Keywords: coumarins; metabolites; biological activity; synthesis; natural sources
1. Introduction
Coumarins are a wide family of secondary metabolites found in various species of plants (more
than 1300 coumarins have been identified from natural sources, especially green plants) but also fungi
and microorganisms [1,2]. The main pathway of coumarin biosynthesis occurs by shikimic acid route,
via cinnamic acid, through phenylalanine metabolism [3]. The history of these natural products began
200 years ago—the name of the class derived from the plant Coumarouna odorata (Dipteryx odorata)
from which the simplest member of this family, coumarin itself (Figure 1), was isolated by Vogel in
1820 [3,4]. Chemically speaking, coumarins are organic heterocycles and their nucleus is represented
by benzo-α-pyrone (2H-1-benzopiran-2-one), whose systematic nomenclature was established by
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 2 of 83
International Union of Pure and Applied Chemistry (IUPAC) [5].
Figure 1. Structure of coumarin nucleus.

Natural
Int. J. Mol. coumarins
Sci. 2020, are subdivided in
different classes based on their www.mdpi.com/journal/ijms
21, 4618; doi:10.3390/ijms21134618 chemical diversity and
complexity—simple coumarins, isocoumarins, furanocoumarins and pyranocoumarins (both
angular and linear), biscoumarins and other coumarins such as phenylcoumarins (Table 1) [6].
Coumarins have several attractive features, such as low molecular weight, simple structure, high
Int. J. Mol. Sci. 2020, 21, 4618 2 of 81

Natural coumarins are subdivided Figure 1.
in 1. Structure of coumarin
different nucleus.
Figure Structureclasses
of coumarin based on their chemical diversity and
nucleus.
Natural coumarins
complexity—simple coumarins,are Figure 1. Structure
subdivided furanocoumarins
isocoumarins, of coumarin
in different classesand nucleus.
based on their chemical
pyranocoumarins (bothdiversity
angular and
Natural
complexity—simplecoumarins are subdivided
coumarins, in different
isocoumarins, classes
furanocoumarins based onandtheir chemical
pyranocoumarins diversity and
and linear), biscoumarins
Natural coumarins and other coumarins
are subdivided such
in as phenylcoumarins
different classes based on (Table
their1) [6].
chemical diversity(bothand
Natural
complexity—simplecoumarins are subdivided
coumarins, and in different
isocoumarins, classes
furanocoumarins based on their chemical
and pyranocoumarins diversity and
(both
angular and
Coumarins linear),
have
complexity—simple biscoumarins
several other coumarins
attractive isocoumarins,
coumarins, features, such
such furanocoumarins
as low as phenylcoumarins
molecular weight,
and simple(Table
pyranocoumarins 1) [6]. (both
structure,
complexity—simple
angular and linear), coumarins, and
biscoumarins isocoumarins,
other coumarins furanocoumarins
such as and pyranocoumarins
phenylcoumarins (Table 1) [6]. (both
Coumarins
high bioavailability,
angular have
high
and linear), several attractive
solubility
biscoumarins in most
and offeatures, such solvents
the coumarins
other organic as low
suchmolecular
asand lowweight,
toxicity,simple
phenylcoumarins which,
(Tablestructure,
1) [6]. high
together
angular and linear),
Coumarins havebiscoumarins
several attractiveand other coumarins
features, as such
such solvents as phenylcoumarins
low molecular weight, (Table
simple 1) [6]. high
structure,
bioavailability,
with their Coumarins
multifaceted high
have solubility in most
several attractive
biological of the
activities,features, organic
ensure them such as a low and low
molecular
prominent roletoxicity,
weight,
as lead which,
simple together
structure,
compounds with
inhigh
Coumarinshigh
bioavailability, havesolubility
several attractive
in most features,
of the such as
organic low molecular
solvents and low weight,
toxicity, simple
which, structure,
together high
with
their multifaceted
bioavailability,
drug research highbiological
and development solubility activities,
in most
[2,6,7]. ofensure
Coumarins them solvents
the organic
exhibit aseveral
prominentand lowroletoxicity,
pharmacologicalas lead effects
compounds
which, together
including in drug
with
bioavailability,
their multifacetedhighbiological
solubilityactivities,
in most ofensure
the organic
them solvents
a prominent and lowroletoxicity,
as lead which,
compounds together with
in drug
research
their and development
multifaceted biological [2,6,7]. Coumarins
activities, ensure exhibit
them a several
prominent
anticoagulant, antimicrobial, anti-inflammatory, neuroprotective, antidiabetic, anticonvulsant and pharmacological
role as lead effects
compounds including
in drug
their multifaceted
research biological [2,6,7].
and development activities, ensure them
Coumarins exhibita prominent role as lead compounds
several pharmacological in drug
effects including
anticoagulant,
research
antiproliferative and antimicrobial,
[8].development
Their importance anti-inflammatory,
[2,6,7].is Coumarins
also clear inexhibitneuroprotective,
the foodseveral
industry antidiabetic,
where theiranticonvulsant
pharmacological and and
effects including
fungicide
research and
anticoagulant, development
antimicrobial, [2,6,7]. Coumarins
anti-inflammatory, exhibit several
neuroprotective, pharmacological
antidiabetic, effects
anticonvulsant includingand
antiproliferative
anticoagulant,
antioxidant [8].investigated
are Their importance
activities antimicrobial, is also clear
anti-inflammatory,
and exploited inMoreover,
the food industry
neuroprotective,
[9,10]. some wherebenzocoumarins
antidiabetic,
natural their fungicide and
anticonvulsant
anticoagulant,
antiproliferative antimicrobial,
[8]. Their anti-inflammatory,
importance is also clear neuroprotective,
in the food antidiabetic,
industry where anticonvulsant
their fungicide and
antioxidant
show anti-algal activities
antiproliferative
activity [8]. are
[11].Theirinvestigated
Their importance
Despite and exploited
is also
being recognized clear [9,10].
in
for their Moreover,
the food industry some natural
where their benzocoumarins
fungicide and
antiproliferative
antioxidant [8].
activities importance
are investigated is also
and clear
exploited in thebiological
[9,10]. food
Moreover,
activities,
industry some wherecoumarins
their
natural
present and
fungicide
benzocoumarins
show
another anti-algal
antioxidant activity
activities
importantactivities [11]. Despite
are investigated
characteristic widely explored being recognized
and exploited for their
[9,10]. Moreover,
by the industry: biological
some natural
their luminescent activities, coumarins
benzocoumarins
properties. The
antioxidant
show anti-algal are investigated
activity Despiteand
[11].characteristic exploited
being recognized[9,10]. forMoreover,
their some natural
biological benzocoumarins
activities, coumarins
presentanti-algal
show
luminescence ofanother
some important
activity
derivatives [11]. Despite
results from being
the widely
recognized
intrinsic explored
charge by the
fortransfer
their industry:
biological
properties their
activities,
of luminescent
coumarins
electron-rich
show
present anti-algal
another activity
important [11].characteristic
Despite beingwidely recognized explored for their
by the biological
industry: activities, coumarins
their luminescent
properties.
present
conjugated The luminescence
another
systems important of some derivatives
characteristic
[3]. These compounds widely
present results from
explored
aresults
broad varietythe
by intrinsic
the chargefor
industry: transfer
their properties
luminescent
presentπ-πanother
properties. The important
luminescence characteristic
of some widely
derivatives explored
from byof
the
applications,
the
intrinsicindustry:
charge theirexample
transfer
as
luminescent
properties
of electron-rich
properties.
photocleavable conjugated
The luminescence
protecting groups or π-π systems
offluorescent
some derivatives [3].
probes These
results
[12]. compounds present a broad
from the intrinsic charge transfer properties variety of
properties.
of The luminescence
electron-rich conjugated of
π-πsome derivatives
systems [3]. results
These from the intrinsic
compounds charge
present a transfer
broad properties
variety of
applications,
of electron-richfor example
conjugated as photocleavable
π-π systems protecting
[3]. Thesegroups or fluorescent
compounds presentprobes
a broad [12]. variety of
of electron-rich
applications, conjugated
for example π-π systems protecting
as photocleavable [3]. Thesegroups compounds presentprobes
or fluorescent a broad [12]. variety of
applications, for exampleTable as photocleavable
1. Classificationprotecting groups or fluorescent probes [12].
of natural coumarins.
applications, for example as photocleavable protecting
Table 1. Classification groups
of natural or fluorescent probes [12].
coumarins.
Class Table
General 1. Classification of natural coumarins.
Structure Examples
Class Table
General 1. Classification of natural coumarins.
Structure
Table 1. Classification of natural coumarins.Examples
Class General Structure Osthole Examplesosteogenic,
(neuroprotective,
Osthole (neuroprotective,
Class
Simple General Structure Examples osteogenic,
Simple
Class General Structure immunomodulatory, anticancer,
Examples
Osthole (neuroprotective, osteogenic,
Simple immunomodulatory, anticancer, osteogenic,
Osthole (neuroprotective, hepatoprotective,
coumarins
coumarins
Simple hepatoprotective,
immunomodulatory, cardiovascular
anticancer,
Osthole (neuroprotective, hepatoprotective,
osteogenic,
coumarins
Simple cardiovascular
immunomodulatory,protective and antimicrobial) [13]
anticancer, hepatoprotective,
coumarins protective and
cardiovascular
immunomodulatory, antimicrobial)
protective [13]
and antimicrobial)
anticancer, [13]
hepatoprotective,
coumarins cardiovascular protective and antimicrobial) [13]
cardiovascular protective and antimicrobial) [13]
Psoralen (antifungal) [14]

Furanocoumarins Psoralen
Psoralen (antifungal)
(antifungal)
Antoghenol [14][15]
[14][14]
(antibacterial)
Psoralen (antifungal)
Furanocoumarins
Furanocoumarins Antoghenol
Psoralen
Antoghenol
Columbianedin (antibacterial)
(antifungal)
(antibacterial) [15]
[14]
[15]
Furanocoumarins Antoghenol(anti-inflammatory)
(antibacterial) [15] [15]
Furanocoumarins Columbianedin (anti-inflammatory)
Columbianedin
Antoghenol (anti-inflammatory) [15][15]
(antibacterial) [15]
Columbianedin (anti-inflammatory) [15]
Columbianedin (anti-inflammatory) [15]
Grandivittin (antibacterial) [16]

Pyranocoumarins Grandivittin (antibacterial) [16]
Pyranocoumarins Grandivittin
Inophyllum A, B, C,(antibacterial)
Grandivittin P, G1, andG
E,(antibacterial) [16]
2 (antiviral) [17]
[16]
Pyranocoumarins
Pyranocoumarins Inophyllum A, B,
Inophyllum A,C,B,E,C,
Grandivittin P,E,GP,1, G
andG
(antibacterial)
1 , andG
2 (antiviral)
[16]
2 [17]
Pyranocoumarins Inophyllum A, B, C, E, P, G1, andG 2 (antiviral) [17]
Inophyllum A,(antiviral)
B, C, E, P,[17]
G1, andG2 (antiviral) [17]
Biscoumarins Dicoumarol (anticoagulant) [18]

Biscoumarins
Biscoumarins Dicoumarol
Dicoumarol (anticoagulant)
(anticoagulant) [18][18]
Int. J. Mol. Sci. 2020, 21, 4618 3 of 81
Table 1. Cont.
Int. J. Mol.
Int.Sci. 2020,
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2020, 21, PEER
x FORREVIEW
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3 of 83
Int. J. Mol.
ClassSci. 2020, 21, x FOR PEER REVIEW
General Structure Examples 3 of 83
Isocoumarins
Isocoumarins
Isocoumarins Thunberginols (antidiabetic)
Thunberginols
Thunberginols [19]
(antidiabetic)
(antidiabetic) [19][19]
Isocoumarins Thunberginols (antidiabetic) [19]
Phenylcoumarins
Phenylcoumarins Isodispar B (anti-inflammatory)
Isodispar [20] [20]
B (anti-inflammatory)
Phenylcoumarins
Phenylcoumarins Isodispar
Isodispar B (anti-inflammatory)
B (anti-inflammatory) [20][20]
This privileged
This privileged scaffold servesserves
scaffold as an as important platform
an important for the
platform fordesign
the designof compound
of compound libraries in in
libraries
This privileged scaffold serves as an important platform for the design of compound libraries in
the search
This for new
the privileged
search fordrug
new
scaffoldcandidates.
drug candidates.
serves asHere, we critically
Here,
an important review
we critically
platform thethe
review
for actual
the state
actual
design of
ofstatetheof art
compound theon artnatural
on natural
libraries
the search for new drug candidates. Here, we critically review the actual state of the art on natural
and
in the and
synthetic
searchsynthetic
coumarins,
for newcoumarins,
drugfocusing focusing
on theonbiological
candidates. the biological
Here, we activity,activity,
critically structure–activity
structure–activity
review the actual relationships
relationships
state art (SARs),
of the(SARs), on
and synthetic coumarins, focusing on the biological activity, structure–activity relationships (SARs),
natural pharmacokinetics
pharmacokinetics
and synthetic (PKs) (PKs)
and their
coumarins, and their potential
potential
focusing applications
applications
on the biological in the in pharma
activity, the pharma and and
structure–activity agri-food
agri-food sectors.
sectors.
relationships
pharmacokinetics (PKs) and their potential applications in the pharma and agri-food sectors.
(SARs), Furthermore,
Furthermore, an overall
pharmacokinetics an overall
overview
(PKs) overview
and theofpotential
oftheir the most
most common common synthetic
synthetic
applications in routes
the routes
pharma appliedapplied
and to obtain
to obtain
agri-food simplesimple
sectors.
Furthermore, an overall overview of the most common synthetic routes applied to obtain simple
coumarins
coumarins
Furthermore, arean are are
provided,provided,
overall together
overview together
with with
a
of thewith a
furtherfurther
mosta common discussion
discussion on
synthetic on on alternative,
alternative,
routes innovative
innovative
applied innovative and
to obtain simple and
greengreen
coumarins provided, together further discussion alternative, and green
synthetic
synthetic
coumarins methodologies.
methodologies.
are provided, together with a further discussion on alternative, innovative and green
synthetic methodologies.
synthetic methodologies.
Metabolism
Metabolism
Metabolism
Metabolism
Coumarin Coumarin is metabolized
is metabolized by cytochrome
by cytochrome P450-linked
P450-linked mono-oxygenase
mono-oxygenase enzyme enzyme (CYP2A6)
(CYP2A6)
Coumarin is metabolized by cytochrome P450-linked mono-oxygenase enzyme (CYP2A6)
system system
Coumarin
in liver inis metabolized
liver microsomes,
microsomes, bywhich which
cytochrome leads
leadsP450-linked to hydroxylation;
mono-oxygenase
to hydroxylation; subsequently,
enzymethe
subsequently, the
(CYP2A6) hydroxylated
system
hydroxylated
system in liver microsomes, which leads to hydroxylation; subsequently, the hydroxylated
in livermetabolite
metabolitemicrosomes,
metabolitefollows follows
phasephase
which
follows leads toII
II conjugation
phase
conjugation
IIhydroxylation;
conjugation
reactions.
subsequently,
reactions.
reactions.
Although
Although thecoumarin
Although
coumarin
hydroxylated
coumarin could could
metabolite
could
potentially
potentiallyfollows
potentially be bebe
phase hydroxylated
II conjugation at
at allreactions.all six possible positions (i.e., carbon atoms 3, 4, 5, 6, 7 and 8) (Figure 1), 7-
hydroxylated
hydroxylated six Although
atsixallpossible positions
possible coumarin
positions couldcarbon
(i.e., (i.e.,
carbon potentially
atoms 3, be
atoms hydroxylated
4, 3,
5, 4,6, 5,7 6,
and at all
8)
7 and 8)six(Figure
(Figure possible
1), 7-
1), 7-
hydroxycoumarin
positions (i.e., carbon atomsand3,3-hydroxycoumarin
4, 5, 6, 7 and 8)are aremain
(Figure the7-hydroxycoumarin
main metabolites. Theandformer one faces phase
II II
hydroxycoumarin
hydroxycoumarin and 3-hydroxycoumarin
and 3-hydroxycoumarin are 1),
the metabolites.
the main The former
metabolites. The 3-hydroxycoumarin
one faces
former one facesphase phase II
are theconjugation
conjugationmain reaction reaction
metabolites. resultingresulting
The in
former the
one
in theinglucuronide glucuronide
faces phase derivative,
derivative,II whereas
conjugation
whereas 3-hydroxycoumarin
reaction
3-hydroxycoumarin resulting in can
be be
the
can can
conjugation reaction resulting the glucuronide derivative, whereas 3-hydroxycoumarin be
furtherfurther
glucuronide metabolized
derivative,
metabolized
further metabolized
by splitting
by whereas
ring ring splitting
by ring3-hydroxycoumarin
to form
splitting
to form two
can products,
two products,
to form two be further
products,
o-hydroxyphenyllactic
metabolized by ring
acid
and and
acidsplitting
acid o- o-
and o-
to form hydroxyphenylacetic
two products, acid
hydroxyphenylacetic acid
(Figure (Figure
o-hydroxyphenyllactic 2) [4,21].
2) [4,21]. acid
Since Since
and the the expression
o-hydroxyphenylacetic
expression of CYP2A6
of CYP2A6 acid (Figure varies
varies between
2) [4,21].
between
hydroxyphenylacetic acid (Figure 2) [4,21]. Since the expression of CYP2A6 varies between
Since individuals,
the
individuals, expression
due to due to genetic
of genetic
CYP2A6 varies
and and environmental
between
environmental individuals,
factors,factors,
due an inter-individual
antointer-individual
genetic and environmental variation
variation in the
factors,
in the
individuals, due to genetic and environmental factors, an inter-individual variation in the
an metabolism
inter-individual
metabolism of of
coumarin coumarin
variation drugs indrugs
the
is is
possible possible
metabolism [4]. [4].
of coumarin drugs is possible [4].
metabolism of coumarin drugs is possible [4].
Phase II metabolism (glucuronide derivative)

HO O O Phase IIPhase
metabolism (glucuronide
II metabolism derivative)
(glucuronide derivative)
HO HO O O O O
7-hydroxycoumarin
7-hydroxycoumarin
7-hydroxycoumarin
O O
O O O O H
OH H H O OH OH OH
OH OH O O H OH OHOH OH OH OH OH
OH OH COOH
H H CHO
O O COOH COOHCOOH
O O CHO CHO
O O O O COOH COOH
O O O O o-hydroxyphenyl- o-hydroxyphenyl- o-hydroxyphenyl-
3-hydroxycoumarin 3,4-epoxide o-hydroxyphenyl-
lactic acid o-hydroxyphenyl-
acetaldehyde o-hydroxyphenyl-
acetic acid
o-hydroxyphenyl-
3-hydroxycoumarin 3,4-epoxide
3,4-epoxide o-hydroxyphenyl- o-hydroxyphenyl-
3-hydroxycoumarin lactic acid
lactic acid acetaldehyde
acetaldehyde acetic acid
acetic acid
Figure 2. Coumarin metabolism.
Figure 2. Coumarin
2.
FigureFigure metabolism.
2. Coumarin
Coumarin metabolism.
metabolism.
In In a recent work, eight coumarin metabolites, which hadbeen

not been identified previously, were
In aa recent
recent work,
In a recent eight
eight coumarin
work,work, eight
coumarin metabolites,
coumarin which
metabolites,
metabolites, which had
had not
which hadbeen
not identified
not been previously,
identified
identified were
previously,
previously, werewere
detected
detected by meansby means of UPLC/Quadrupole-TOF tandem mass spectrometry in human urine [22].
detected
detected by by of
means UPLC/Quadrupole-TOF
means
of of UPLC/Quadrupole-TOF
UPLC/Quadrupole-TOF tandem mass
tandem spectrometry
tandem
mass mass in human
spectrometry
spectrometry in urine [22].
in human
human Among
urineurine
[22].[22].
them, Among
positional them, positional
isomers of isomers of 7-hydroxycoumarin
7-hydroxycoumarin glucuronide and glucuronide and 7-hydroxycoumarin
7-hydroxycoumarin sulphate were
Among Among them,
them, were positional
positional isomers isomers of 7-hydroxycoumarin
of 7-hydroxycoumarin glucuronide
glucuronide and 7-hydroxycoumarin
andsubstituent
7-hydroxycoumarin
found. sulphate
It was proposed found.
that It wasisomers
such proposed that bear
should such the
isomers shouldinbear
substituent the
position 5, 6 orin in position 5, 6
8. Metabolites
sulphate
sulphate were found. It was proposed that such isomers should bear the substituent in position
5, 6 5, 6
or 8. Metabolites coming from a double hydroxylation and subsequent conjugation position
were found. It was proposed that such isomers should bear the substituent with glucuronic
coming or from
8.
or 8. Metabolitesa double
Metabolites
coming hydroxylation
coming
from(andfrom
a doublea and
doublesubsequent conjugation
hydroxylation
hydroxylation and with
subsequent
and subsequent glucuronic acid
conjugation or
with sulphate
glucuronic
acid or sulphate group their isomers) were detected as well. conjugation with glucuronic
Another metabolite was the one
group (and
acid ortheir
acid orobtained
sulphate isomers)
sulphate
group group
(andwere(anddetected
their their aswere
well.were
isomers)
isomers) Another metabolite
detected
detected as as well.
well. wasmetabolite
the metabolite
Another
Another one obtained theby
was was thea one
one
by a double hydroxylation of the coumarin ring, followed by methylation and
double hydroxylation
obtained
obtained by a of the
double coumarin
by a doubleat hydroxylation ring,
hydroxylation followed
of theby methylation
coumarin and
ring, glucuronidation
followed by at the
methylation two
and and
glucuronidation the two newlyofgenerated the coumarin ring,groups.
hydroxyl followed by methylation
Finally, the N-acetylcysteine
newly glucuronidationat theat two
generated hydroxyl
glucuronidation the newly
groups.twoFinally,
newly generated
the
generated hydroxyl
N-acetylcysteine groups.
conjugated Finally,
metabolitethewasN-acetylcysteine
identified.
conjugated metabolite was identified. In this hydroxyl groups. Finally,
work, o-hydroxyphenylacetic the
acidN-acetylcysteine
was also detected in
conjugated
conjugated metabolite
metabolite was
was identified.identified.
In thisIn this work, o-hydroxyphenylacetic acid was also detected
in in
the samples, whereas free coumarin andwork, o-hydroxyphenylacetic
o-coumaric acid were not found, acid indicating
was also detected
that coumarin
the
the samples, samples, whereas
whereas metabolizedfree
free coumarin coumarin and o-coumaric
and excretion,
o-coumaricafter acid
acidoral
were were not found, indicating
not found, indicating that
thatthat coumarin
coumarin
was completely before administration. This meant o-coumaric
was completely
was completely metabolized
metabolized beforebefore excretion,
excretion, after after oral administration.
oral administration. This This
meantmeant
that that o-coumaric
o-coumaric
Int. J. Mol. Sci. 2020, 21, 4618 4 of 81
In this
Int. work,
J. Mol. o-hydroxyphenylacetic
Sci. 2020, 21, x FOR PEER REVIEWacid was also detected in the samples, whereas free coumarin4and
of 83
o-coumaric acid were not found, indicating that coumarin was completely metabolized before excretion,
acid oral
after found in human plasma
administration. [23] underwent
This meant a biotransformation
that o-coumaric processplasma
acid found in human before [23]
being eliminated,
underwent a
probably leading to o-hydroxyphenylacetic acid.
biotransformation process before being eliminated, probably leading to o-hydroxyphenylacetic acid.
2.2.Biological
BiologicalActivities
Activities
2.1.
2.1.Antioxidant
AntioxidantActivity
Activity
In
Inaahealthy
healthyhuman
humanbody, body,normal
normalmetabolic
metabolicprocesses
processesproduce
producefree
freeradicals
radicalsand
andother
otherhighly
highly
reactive
reactivespecies
speciessuch
suchasasions,
ions,molecules
molecules with
with unpaired
unpaired electrons, Reactive
electrons, ReactiveOxygen,
Oxygen,Carbon, Nitrogen
Carbon, or
Nitrogen
Sulfur Species (ROS, RCS, RNS or RSS). When these species are overproduced, oxidative
or Sulfur Species (ROS, RCS, RNS or RSS). When these species are overproduced, oxidative processes processes might
cause
mightcellular
cause damage,
cellular affecting
damage, cellular components
affecting and causing ionic
cellular components imbalance
and causing or mitochondrial
ionic imbalance or
disfunction
mitochondrial[24].disfunction
The role of[24].
oxidative
The rolestress in different
of oxidative pathologies
stress is well
in different know: inflammation,
pathologies is well know:
cardiovascular
inflammation, diseases, cancer,diseases,
cardiovascular diabetes cancer,
and even neurodegenerative
diabetes disorders often count
and even neurodegenerative oxidative
disorders often
damage among theirdamage
count oxidative pathological
amongfeatures
their[25–27]. Therefore,
pathological exogenous
features antioxidants
[25–27]. mightexogenous
Therefore, be useful
in order to maintain
antioxidants might be theuseful
right concentration of radicals,
in order to maintain reducing
the right the amounts
concentration of free radicals
of radicals, reducingandthe
avoiding
amountsoxidative stress [28].
of free radicals and avoiding oxidative stress [28].
The
Theantioxidant
antioxidantpotential
potentialofof
natural and
natural andsynthetic
synthetic coumarins
coumarins hashas
been deeply
been investigated
deeply in thein
investigated
last years and it became clear that poly-hydroxy or phenolic coumarins are efficient
the last years and it became clear that poly-hydroxy or phenolic coumarins are efficient antioxidants antioxidants in
biologicals systems
in biologicals [29].[29].
systems Here below
Here the the
below most recent
most updates
recent in this
updates fieldfield
in this are are
reported.
reported.
In
In 2019, Couttolenc
Couttolencand and collaborators
collaborators studied
studied the radical
the radical scavenging
scavenging activityactivity
of three of three
hydroxy-
hydroxy-4-methylcoumarins (1–3, Figure 3) by means of experimental and
4-methylcoumarins (1–3, Figure 3) by means of experimental and theoretical methods [30]. theoretical methods [30].
Figure 3. Coumarin structures reported by Couttolenc et al. [30].

Figure 3. Coumarin structures reported by Couttolenc et al. [30].
Firstly, the
Firstly, the scavenging
scavenging activity
activity of of the
the compounds
compoundswas wasevaluated
evaluatedononABTS ABTS (2,2′-azino-bis
(2,20 -azino-bis (3-
ethylbenzothiazoline-6-sulfonic acid) diammonium salt), DPPH (2,2-diphenyl-1-picrylhydrazyl)
(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt), DPPH (2,2-diphenyl-1-picrylhydrazyl) and
galvinoxyl
and galvinoxyl radicals
radicalsas Trolox
as Trolox (a Vitamin
(a Vitamin E analogue)
E analogue) equivalent
equivalent antioxidant
antioxidantcapability
capability(TEAC).
(TEAC).The
results showed that, whereas 1 did not exhibit radical scavenging activity,
The results showed that, whereas 1 did not exhibit radical scavenging activity, 2 resulted more active 2 resulted more active than
Trolox
than against
Trolox the ABTS•+
against the ABTS•+ radical (EC50(EC
radical 30.83 μM) and 3 displayed better antioxidant activity than
50 30.83 µM) and 3 displayed better antioxidant activity
Trolox
than against
Trolox againstABTS•+,
ABTS•+, DPPH DPPH and andgalvinoxyl
galvinoxyl radicals
radicals(EC(EC50 values of 39.98, 150.99 and 13.19 μM,
50 values of 39.98, 150.99 and 13.19 µM,
respectively). It is likely that such differences in antioxidant
respectively). It is likely that such differences in antioxidant activity may activity mayrelyrelyononthethedifferences
differencesinin
therelative
the relativepositions
positionsofofhydroxy
hydroxygroups groups[31].
[31].Then,
Then,compound
compound3,3,which whichshowed
showedthe thebest
bestscavenging
scavenging
activity, was evaluated for its primary antioxidant capacity. In this step,
activity, was evaluated for its primary antioxidant capacity. In this step, three reaction mechanisms three reaction mechanisms
wereconsidered:
were considered:single singleelectron
electrontransfer
transfer(SET),
(SET),hydrogen
hydrogentransfer
transfer(HT)(HT)and
andradical
radicaladduct
adductformation
formation
(RAF),involving
(RAF), involving•OOH, •OOH, •OOCH
•OOCH 3 and •CH(OH)CH3 as radicals. These experiments were carried
3 and •CH(OH)CH3 as radicals. These experiments were carried out
out in lipidic and aqueous
in lipidic and aqueous media, in order media, in order to mimic
to mimic membrane
membrane and intra-cellular
and intra-cellular environment
environment [32],
[32], [33].
[33]. The results indicated that different mechanisms are involved
The results indicated that different mechanisms are involved depending by the medium and that depending by the medium and
that positions
positions 4, 7 and4, 78 and 8 of compound
of compound 3 are probably
3 are probably involvedinvolved in HT mechanism,
in HT mechanism, whereas whereas
positions positions
3, 4, 5
and 7 could be involved in RAF mechanism. Finally, the secondary antioxidant activity of compound of
3, 4, 5 and 7 could be involved in RAF mechanism. Finally, the secondary antioxidant activity 3
incompound
aqueous media 3 in aqueous media at pH
at physiological physiological
was evaluated. pH was evaluated.
In the In thereaction
Haber-Weiss Haber-Weiss reaction
(i.e., the (i.e.,
reduction
ofthe reduction
copper in aqueousof copper
media in andaqueous
subsequent media and subsequent
copper-catalyzed copper-catalyzed
hydroxy radical formation),hydroxy 3 was radical
able
formation), 3 was able to inhibit copper (II) reduction avoiding oxidative
to inhibit copper (II) reduction avoiding oxidative stress. It was found that hydroxy groups in position stress. It was found that
7hydroxy
and 8 aregroups
fundamentalin position
for the7 primary
and 8 are andfundamental for the primary
secondary antioxidant activityand secondary
in both lipid and antioxidant
aqueous
activity in both lipid and aqueous media. Recently, Wang and
media. Recently, Wang and co-workers investigated the antioxidant activity and the mechanism of the co-workers investigated the
antioxidant activity and the mechanism of the antiradical action of six coumarin-fused coumarins (4–
9, Figure 4) previously synthesized by Xi and Liu [34,35].
Int. J. Mol. Sci. 2020, 21, 4618 5 of 81
antiradical action
Int. J. Mol. Sci. 2020,of
21,six coumarin-fused
x FOR PEER REVIEW coumarins (4–9, Figure 4) previously synthesized by Xi and
5 of 83
Liu [34,35].
Figure 4.
Figure Chemical structure
4. Chemical structure of of
coumarin-fused
coumarin-fused coumarins
coumarins4–9. 4–9.
Figure 4. Chemical structure of coumarin-fused coumarins 4–9.
Density
Density functional
functional theory
theory (DFT)
(DFT) calculations
calculations were
were performed,
performed, followed
followed byby
thethe examination
examination of of
thethe
Density functional theory (DFT) calculations were performed, followed by the examination of the
primary
primary mechanisms,
mechanisms, including
including HT,HT, electron
electron transfer-proton
transfer-proton transfer
transfer (SET-PT)
(SET-PT) and and sequential
sequential proton
proton
primary mechanisms, including HT, electron transfer-proton transfer (SET-PT) and sequential proton
loss
loss transfer
transfer (SPLET).
(SPLET). TheThe most
most stable
stable conformation
conformation of of
allall
thethe compounds
compounds waswas a non-planar
a non-planar structure,
structure,
loss transfer (SPLET). The most stable conformation of all the compounds was a non-planar structure,
duedueto to
thethe steric
steric repulsion
repulsion of of
thethe groups
groups in positions
in positions 5 and
5 and 50 . 5′.
Such Such conformation
conformation waswas retained
retained bybythethe
due to the steric repulsion of the groups in positions − , ArOH5 and +•5′. Such conformation was retained by the
correspondent
correspondent anions
anions and and cation
cation radicals
radicals (ArO
(ArO −, ArOH +•).
). HT HTprocess
process resulted
resulted thethe most
most significant
significant in in
correspondent anions and cation radicals (ArO−, ArOH+•). HT process resulted the most significant in
gasgas
or or non-polar
non-polar phase,
phase, where
where compound
compound 9 showed
9 showed thethe highest
highest activity.
activity. TheTheHTHT path
path waswas possible
possible forfor
gas or non-polar phase, where compound 9 showed the highest activity. The HT path was possible for
compounds
compounds 7, 87,and
8 and 9, having
9, having twotwoor or three
three OH OH groups,
groups, whereas
whereas 4 resulted
4 resulted inactive
inactive duedue
to to
thethe absence
absence
compounds 7, 8 and 9, having two or three OH groups, whereas 4 resulted inactive due to the absence
of of
OH OH groups;
groups; compound
compound 5, with
5, with only
only thethe
6-OH6-OH group,
group, was was lessless active
active than
than other
other compounds
compounds andand6 6
of OH groups; compound 5, with only the 6-OH group, was less active than other compounds and 6
could
could merely
merely traptrap DPPH
DPPH radical
radical withwith a small
a small raterate constant.
constant. AA second
second HAT HAT process
process waswas possible
possible only
only
could merely trap DPPH radical with a small rate constant. A second HAT process was possible only
forfor compound
compound 9 and
9 and thisthis finding
finding could
could explain
explain thethe higher
higher activity
activity of of
this this molecule.
molecule. InIn polar
polar media,
media,
for compound 9 and this finding could explain the higher activity of this molecule. In polar media,
SPLET
SPLET mechanism
mechanism was was favored—in
favored—in thisthis case
case thethe studied
studied compounds
compounds werewere more
more efficient
efficient than
than Trolox
Trolox
SPLET mechanism was favored—in this case the studied compounds were more efficient than Trolox
in in
thethe deprotonationstep
deprotonation stepand,
and,among
among them, compound compound77resulted resultedtotobebe thethemost
mostpromising,
promising, being more
being
in the deprotonation step and, among them, compound 7 resulted to be the most promising, being more
prone
more thanthan
prone the the
other compounds
other compounds to deprotonate
to deprotonate in a polar environment.
in a polar environment.
prone than the other compounds to deprotonate in a polar environment.
TheThe antioxidant
antioxidant potential
potential of of coumarin
coumarin nucleus
nucleus cancanbebe exploited
exploited in in
thethe production
production of of
newnew hybrid
hybrid
The antioxidant potential of coumarin nucleus can be exploited in the production of new hybrid
compound
compound with
with enhanced
enhanced antioxidant
antioxidant activity.
activity. A recent
A recent example
example of of this
this strategy
strategy is the
is the synthesis
synthesis of of
a a
compound with enhanced antioxidant activity. A recent example of this strategy is the synthesis of a
new series of chitosan derivatives (10a–d, Figure 5) containing the coumarin nucleus, achieved by Li Li
new series of chitosan derivatives (10a–d, Figure 5) containing the coumarin nucleus, achieved by
new series of chitosan derivatives (10a–d, Figure 5) containing the coumarin nucleus, achieved by Li
andand collaborators
collaborators [36].
[36].
and collaborators [36].
Figure
Figure 5. Chitosan
5. Chitosan derivatives
derivatives 10a–d.
10a–d.
Figure 5. Chitosan derivatives 10a–d.
TheThe antioxidant
antioxidant potential
potential of of compounds
compounds 10a–d
10a–d waswas investigated
investigated byby evaluating
evaluating thethe inhibition
inhibition of of
The antioxidant potential of compounds 10a–d was investigated by evaluating the inhibition of
lipid
lipid peroxidation,
peroxidation, metal
metal ionion chelation
chelation and
and free-radical
free-radical scavenging
scavenging activity.
activity. Since
Since both
both chitosan
chitosan and
and
lipid peroxidation, metal ion chelation and free-radical scavenging activity. Since both chitosan and
coumarins have antioxidant properties themselves, the synthesized compounds were expected to to
coumarins have antioxidant properties themselves, the synthesized compounds were expected
coumarins have antioxidant properties themselves, the synthesized compounds were expected to
show
show a stronger
a stronger activity.
activity. Lipid
Lipid peroxidation
peroxidation inhibition
inhibition was
was determined
determined byby quantifying
quantifying thiobarbituric
thiobarbituric
show a stronger activity. Lipid peroxidation inhibition was determined by quantifying thiobarbituric
acid-reactive substance (TBARS), using linoleic acid as a reference compound [37]. The results
displayed the ability of the synthesized molecules to inhibit TABRS in a concentration-dependent
manner; compound 10d emerged as the most active (IC50 = 0.11 mg/mL), showing a more efficient
manner; compound 10d emerged as the most active (IC50 = 0.11 mg/mL), showing a more efficient
scavenging activity than chitosan alone (IC50 = 0.38 mg/mL). Then, the radical-scavenging activity was
scavenging activity than chitosan alone (IC50 = 0.38 mg/mL). Then, the radical-scavenging activity was
Int. J. Mol. Sci. 2020, 21, 4618 6 of 81
Int. manner; compound
J. Mol. Sci. 2020, 21, x FOR10d
PEER REVIEW as the most active (IC50 = 0.11 mg/mL), showing a more efficient
emerged 6 of 83
scavenging activity than chitosan alone (IC50 = 0.38 mg/mL). Then, the radical-scavenging activity
evaluated.
was evaluated.For thisFor purpose, free radicals
this purpose, •OH,•OH,
free radicals DPPH DPPHandand •− were
O2•O− 2were used. Compounds
used. Compounds 10a-d
10a–d
showed a stronger •OH scavenging activity (IC50 = 0.09–0.12 mg/mL) compared to that of chitosan.
showed a stronger •OH scavenging activity (IC 50 = 0.09-0.12 mg/mL) compared to that of chitosan.
These results
These resultssuggested
suggested thatthat
thethe
coumarin
coumarin moiety
moietystrongly enhances
strongly enhanceschitosan antioxidant
chitosan properties.
antioxidant properties.
Since the chelating ability of antioxidants prevents oxidative stress by avoiding
Since the chelating ability of antioxidants prevents oxidative stress by avoiding •OH production, •OH production,
iron-chelating
iron-chelating properties
properties of of
thethe
new newcompounds
compounds were also
were evaluated
also evaluated through
through measurements
measurements of of
inhibition
inhibition of of
ferrozine-Fe
ferrozine-Fe 2+ complex
2+ complex formation.
formation.Again,
Again,thethe
obtained
obtained results (IC(IC
results =
50 = 0.02-0.04
50 mg/mL)
0.02–0.04 mg/mL)
werewere better
betterthanthanthose of of
those chitosan
chitosanalone.
alone.Finally, cytotoxicity
Finally, cytotoxicityof of
thethe
synthesized
synthesized compounds
compounds was was
tested: no no
tested: toxic effects
toxic were
effects were detected
detected onon3T3-L1
3T3-L1andandHHL-5
HHL-5cells.
cells.Interestingly,
Interestingly, 3T3-L1
3T3-L1 cells showed an
an increase
increase in in their
their viability,
viability,probably
probablybecause
becauseof ofthe
theantioxidant
antioxidantactivity
activityof ofthe
thetested
testedcompounds.
compounds.
A similar approach was followed by Popova and co-workers,
A similar approach was followed by Popova and co-workers, who designed and who designed and synthesized a
synthesized
series of 4-methylcoumarins
a series of 4-methylcoumarins withwith
tert-butyl, isobornyl
tert-butyl, andand
isobornyl isocamphyl
isocamphyl substituents (11,(11,
substituents 12, 12,
13–17,
13–17,
Figure 6) [38].
Figure 6) [38].
Figure
Figure 6. Chemical
6. Chemical structures
structures of tert-butylcoumarins
of tert-butylcoumarins 11 and
11 and 12 and
12 and terpenylcoumarins
terpenylcoumarins 13–17.
13–17.
TheThe synthesizedcompounds
synthesized compoundswere were evaluated
evaluated ininterms
termsofof antioxidant, membrane-protective
antioxidant, membrane-protective (MPA)
and radical-scavenging (RSA) activities in vitro. All the tested compounds, at a concentration
(MPA) and radical-scavenging (RSA) activities in vitro. All the tested compounds, at a concentration of 100
µM,μM,
of 100 exhibited goodgood
exhibited inhibitory activity
inhibitory against
activity lipidlipid
against peroxidation products
peroxidation (LPO)
products formation
(LPO) (IC50 =
formation
3.33–7.12 nM), whereas 7-hydroxy-4-methylcoumarin, used as reference compound,
(IC50 = 3.33 – 7.12 nM), whereas 7-hydroxy-4-methylcoumarin, used as reference compound, showed showed no activity.
no The scavenging
activity. activity, evaluated
The scavenging using DPPH,
activity, evaluated usingstrictly
DPPH,depended on the structure:
strictly depended only isobornyl
on the structure: only
derivatives
isobornyl showedshowed
derivatives moderate activity in
moderate the DPPH
activity in theassay
DPPH (compound 15 showed
assay (compound 15RSA%
showed= 57.48
RSA% ± =0.60
at 100 µM; RSA% = 87.95 ± 0.22 at 500 µM). Moreover, the protective activity towards
57.48 ± 0.60 at 100 μM; RSA% = 87.95 ± 0.22 at 500 μM). Moreover, the protective activity towards cell cell membrane
was assessed,
membrane measuring
was assessed, the inhibitory
measuring activity against
the inhibitory activityHagainst
2 O2 -induced hemolysis
H2O2-induced of red blood
hemolysis cells
of red
(RBCs). In all the experiments, the most promising compound was 15, having
blood cells (RBCs). In all the experiments, the most promising compound was 15, having two two isobornyl moieties.
isobornylFrom these studies, it appears clear that coumarin derivatives show a great potential as antioxidant,
moieties.
membrane-protective
From these studies,and radical-scavenging
it appears clear that compounds and that their
coumarin derivatives activity
show depends
a great mainly
potential as on
the number and the position of the hydroxy groups.
antioxidant, membrane-protective and radical-scavenging compounds and that their activity
depends mainly on the number and the position of the hydroxy groups.
2.2. Anticancer Activity
The term
2.2. Anticancer “cancer” defines a wide range of diseases caused by the accumulation of mutations and
Activity
characterized by a multi-step process, involving many different factors which may not directly cause
The term “cancer” defines a wide range of diseases caused by the accumulation of mutations
cancer themselves but can increase the chances of genetic mutations [39,40].
and characterized by a multi-step process, involving many different factors which may not directly
Recently, Maleki et al. have synthesized eighteen O-prenylated coumarin derivatives and tested
cause cancer themselves but can increase the chances of genetic mutations [39,40].
them on HeLa cervical cancer and HDF normal cells by MTT assay [41]. The most promising compounds
Recently, Maleki et al. have synthesized eighteen O-prenylated coumarin derivatives and tested
are reported in Figure 7.
them on HeLa cervical cancer and HDF normal cells by MTT assay [41]. The most promising
compounds are reported in Figure 7.
Int. J. Mol. Sci. 2020, 21, 4618 7 of 81

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW O 7 of 83
O
O O O O O
O
5-geranyloxycoumarin (5-GC) 6-geranyloxycoumarin (6-GC)
O O O O
5-geranyloxycoumarin (5-GC) 6-geranyloxycoumarin (6-GC)

O O O
O O
O O O
6-farnesyloxycoumarin (6-FC) 8-geranyloxycoumarin (8-GC)
Figure 7. Structure of theOmostOactive O-prenylated coumarins.

6-farnesyloxycoumarin (6-FC) 8-geranyloxycoumarin (8-GC)
The results represent a good starting point for the design of novel derivatives, because most of
the examined compounds Figure
Figure 7.7. Structureselective
exhibited of the mosttoxicity O-prenylated
active on HeLa cellscoumarins.
O-prenylated coumarins.
(IC50 values between 136.4 ± 1.90
μM and 172.2 ± 1.80 μM after 24 h), whereas no negative effects on HDF normal cell’s growth was
The
The results
results represent
represent aagood
good starting
startingpoint forfor
point thethe
design of novel derivatives, because most of the
detected. SAR studies proved that the substitution on design of novel
C6 position derivatives,
of coumarin because
nucleus most
provided of
the
examined
the examinedcompounds
compounds exhibited
exhibitedselective toxicity
selective on HeLa
toxicity on cells (IC
HeLa cells values
50 (IC between
50 values between ± 1.90
136.4136.4 ± µM
1.90
best anticancer activity, followed by substitution on C8. In addition, it was found that the cytotoxic
and
μM and ± 1.80
172.2172.2 µMμM after 24 24 h),h),
whereas nononegative effects ononHDFHDFnormal cell’s growth was
properties of ±O-prenylated
1.80 after coumarins whereas
depends negative
on effects
the length of the prenyl normal
chain, cell’s
whichgrowth was
increases the
detected.
detected. SAR
SAR studies
studies proved
proved that
that the
the substitution
substitution on
on C6
C6 position
position of
of coumarin
coumarin nucleus
nucleus provided
provided the
the
lipophilicity of the molecule, thus facilitating its penetration into the cells.
best
best anticancer
anticancer activity,
activity, followed
followed by
by substitution
substitution on on C8.
C8. In
In addition,
addition, it
it was
was found that
that the cytotoxic
The cytotoxic activity of prenylated-coumarin derivatives had beenfound
evaluated the cytotoxic
also by other
properties
properties of O-prenylated
of O-prenylated coumarins
coumarins depends on the length of the prenyl chain, which increases
increases the
groups. Recent studies have showndepends on therole
a functional length of the prenyl chain,
of lipoxygenases (LOXs)which the
in carcinogenesis,
lipophilicity
lipophilicity of
of the
the molecule,
molecule, thus
thus facilitating
facilitating its
its penetration
penetration into
into the
the cells.
cells.
precisely in prostatic cancer and the capability of 5- farnesyloxycoumarin (18, Figure 8) to inhibit 15-
The
The cytotoxic
cytotoxicactivity
activityof of
prenylated-coumarin
prenylated-coumarin derivatives
derivativeshad been
had evaluated also byalso
been evaluated otherbygroups.
other
LOX-1 [42–44]
Recent studies have shown a functional role of lipoxygenases (LOXs)
groups. Recent studies have shown a functional role of lipoxygenases (LOXs) in carcinogenesis, in carcinogenesis, precisely in
prostatic
preciselycancer and the
in prostatic capability
cancer and the of 5- farnesyloxycoumarin
capability (18, Figure 8) to
of 5- farnesyloxycoumarin inhibit
(18, 15-LOX-1
Figure [42–44].
8) to inhibit 15-
LOX-1 [42–44]
Figure 8. Chemical structure of 5-farnesyloxycoumarin (18) and 8-farnesyloxycoumarin (19).

Figure 8. Chemical structure of 5-farnesyloxycoumarin (18) and 8-farnesyloxycoumarin (19).
Starting from these observations, Orafaie and collaborators investigated the inhibitory activity
Starting18
of compound from
on these
15-LOX-1observations,
[45]. TheOrafaie
cytotoxic andeffects
collaborators
of 18 were investigated
evaluatedthe byinhibitory
means of activity
MTT
of Figure 8.18Chemical
compound on structure
15-LOX-1 ofThe
[45]. 5-farnesyloxycoumarin
cytotoxic effects of(18)
18 and
were 8-farnesyloxycoumarin
evaluated by means (19).
of 4-MMPB
MTT assay
assay on two carcinoma cell lines (PC-3 and DU145) and a normal cell line (HFF3), using
on two carcinoma cell lines (PC-3
(4-methyl-2-(4-methylpiperazinyl) and DU145)
pyrimido [4,5-b] and a normal cellasline
benzothiazine) (HFF3),compound.
reference using 4-MMPB When(4-
Starting from these observations,
methyl-2-(4-methylpiperazinyl) Orafaie
pyrimido and benzothiazine)
[4,5-b] collaborators investigated
as reference the inhibitoryWhen
compound. activityPC3
PC3
ofand and DU145
compound human
18 on PCa cells
15-LOX-1 [45]. were
The treated with
cytotoxic effectsdifferent
of 18 concentrations
were evaluated by ofmeans
both 4-MMPB
of MTT and
assay
18 for DU145
24,carcinomahuman
48 and 72cell PCa cells
h, alines were
dose-dependent treated with different
and time-dependent concentrations
decrease of both 4-MMPB
in the survival and 18
of the cells for
on24,two48 and 72PC3 h, acells (PC-3
dose-dependent and DU145) and a normal cell line (HFF3), using 4-MMPB (4-
was exhibited.
methyl-2-(4-methylpiperazinyl) resulted to beand
pyrimido more time-dependent
sensitive
[4,5-b] thandecrease
benzothiazine) DU145 in the
cells
as reference
survival
to both of the (IC
inhibitors
compound. When
cells in
50
PC3
was
exhibited.
µg/mL PC3
for compound cells resulted
on PC3
18 cells to be
cells:more sensitive
24 h, 40.1 ± than DU145
7.9, 48 h, cells to both
27.05 ± 3.9, 72ofh,both inhibitors
26.434-MMPB (IC
± 2.1; DU145 50 in μg/mL
cells:
and
forDU145
compound human 18 PCa
on PC3 were
cells: 24treated
h, 40.1with± 7.9,different
48 h, 27.05concentrations
± 3.9, 72 h, 26.43 ± 2.1; DU145and 18
cells: forh,
24
24 h,
24,98.1498.14
48 and ± 48.3,
72 48h, h, 48 h, 62.5
a dose-dependent± 17.3, 72 h, 41.85 ±
and time-dependent 7.8; IC 50 in µg/mL
decrease for 4-MMPB:
in the survival PC3
of24the cells:
cells24was h,
32.01 ±
±3.2,
48.3,
48 h,
62.5±±1.9,
25.47
17.3,
72
72
h,
h, 41.85
18.97 ±
±2.8;
7.8;DU145
IC50 incells:
μg/mL 24
for
h,
4-MMPB:
35.22 ± 1.9,
PC3
48
cells:
h, 27.84 ±
h,2.22,
32.0172 ± h,
3.2,
exhibited.
48 h, 25.47 PC3± cells72resulted
1.9, h, 18.97 to± be more
2.8; DU145 sensitive
cells: than
24 h, DU145
35.22 ± cells
1.9, 48 toh,both
27.84 inhibitors
± 2.22, 72(IC
h, 50 in μg/mL
19.52 ± 4.92).
19.52 ± 4.92). Moreover,
forMoreover,
compound 18 on PC3 compound h,18 had no significant
48 h, 27.05anti-proliferative activity on normalcells: cells.
compound 18cells:
had no 24 significant
40.1 ± 7.9,anti-proliferative ± 3.9, 72 h, on
activity 26.43 ± 2.1;cells.
normal DU145 Concerning 24 h, the
Concerning
98.14 ± 48.3, the
48 mechanism
h, 62.5 ± of 72
17.3, action,
h, it was
41.85 ± found
7.8; IC that 5- farnesyloxycoumarin acts as a cytotoxic agent
50 in μg/mL for 4-MMPB: PC3 cells: 24 h, 32.01 ± 3.2,
mechanism of action, it was found that 5- farnesyloxycoumarin acts as a cytotoxic agent causing
causing
48chromatin
h, 25.47chromatin
1.9, 72condensation
± condensation
h, 18.97and ± 2.8; and
DU145DNA damage
cells: 24 h, and induces
35.22 ±the 48the
1.9,arresth,ofarrest
27.84 ±of2.22,
the cell h,cycle
72 in 19.52 0 /G1
in±G4.92).
DNA damage and induces the cell cycle G 0/G1 phase. A
phase.
Moreover,A similar
compound study 18washadcarried
no out on 8-farnesyloxycoumarin
significant anti-proliferative activity (19, onFigure
normal 8) by Hosseinymehr
cells. Concerningand the
similar study was carried out on 8-farnesyloxycoumarin (19, Figure 8) by Hosseinymehr and
collaborators
mechanism of[46]. Again,
action, it the
was coumarin
found derivative
that 5- showed inhibitory
farnesyloxycoumarin activity
acts as a on 15-LOX-1
cytotoxic in PC3
agent and
causing
collaborators [46]. Again, the coumarin derivative showed inhibitory activity on 15-LOX-1 in PC3
DU145
chromatin cell lines, thus inducingDNA
condensation apoptosis of theandcancer cell, with the same mechanism inofGcompound 18.
and DU145 cell lines, andthus inducing damage apoptosis induces
of thethecancer
arrest of the
cell, cell the
with cyclesame 0/G1 phase. A
mechanism of
PC3 cellsstudy
similar resultedwas to be more out
carried sensitive to 19 inhibition than DU145
on 8-farnesyloxycoumarin (19, cells;
Figure IC508) values of 19 on PC3 cells
by Hosseinymehr and
collaborators [46]. Again, the coumarin derivative showed inhibitory activity on 15-LOX-1 in PC3
and DU145 cell lines, thus inducing apoptosis of the cancer cell, with the same mechanism of
compound 18. PC3 cells resulted to be more sensitive to 19 inhibition than DU145 cells; IC50 values of
Int. J. Mol. Sci.
compound 2020,
18. PC3 21, 4618 resulted to be more sensitive to 19 inhibition than DU145 cells; IC50 values 8 of 81
19 on PC3 cells cells
were similar to those of cisplatinum (PC3 IC50 in μg/mL 24 h, 34.98 ± 95.57, 48 h,of14.92
19 on± PC3
40.76,cells were
72 h, 14.12similar to those of cisplatinum (PC3 IC50 in μg/mL 24 h, 34.98 ± 95.57, 48 h, 14.92
± 38.58).
± 40.76, 72Halawa
h, 14.12et ± 38.58).
al.of synthesized and characterized
were similar to those cisplatinum (PC3 IC50 in µg/mL a24new series
h, 34.98 of 4-arylamino-3-nitrocoumarin
± 95.57, 48 h, 14.92 ± 40.76, 72 h,
Halawa
analogues et al. synthesized and characterized a new series of 4-arylamino-3-nitrocoumarin
14.12 ± 38.58). from 4-hydroxycoumarin and tested them on the human cervix carcinoma cell line KB-3-
analogues[47]. from
1 Halawa These 4-hydroxycoumarin
compounds wereandand tested
found them
to target theon the human cervix carcinoma cell lineI)KB-3-
et al. synthesized characterized aDNA-Topo
new seriesI of (human Topoisomerase
4-arylamino-3-nitrocoumarin complex,
1 [47].
thusThese compounds
blocking were found to target
cell replication leading thetoDNA-Topo
cell I (human Topoisomerase I) complex,
analogues from 4-hydroxycoumarin and tested them on thedeath.
humanAmong this series,
cervix carcinoma thiazolidinylidene
cell line KB-3-1 [47].
thusderivative
blocking 20 cell(Figure
replication and leading to cell death. Among this the
series,
bestthiazolidinylidene
These compounds were 9) containing
found a malononitrile
to target the DNA-Topo fragment
I (humanexhibited
Topoisomerase cytotoxic activity
I) complex, thus with
derivative
an ICcell20 (Figure
50 value
9) containing
of 21 andμM.leadinga malononitrile
The cytotoxicity fragment exhibited the best cytotoxic activity with
blocking replication to cell death.ofAmong
20 wasthisexplained also by dockingderivative
series, thiazolidinylidene studies which
20
an IC 50 value of 21 μM. The cytotoxicity of 20 was explained also by docking studies which
highlighted that a itmalononitrile
forms important H-bonds
(Figure 9) containing fragment exhibitedwith Arg364,
the best Asp533,
cytotoxic activityGln633
with anand 5′-thio-2′
IC50 value
highlighted that it phosphonic
deoxyguanosine forms important H-bonds
acidexplained
of the DNA with Arg364, Asp533, Gln633 and 5′-thio-2′
of 21 µM. The cytotoxicity of 20 was alsobackbone.
by docking studies which highlighted that it forms
deoxyguanosine phosphonic acid of the DNA backbone.0
important H-bonds with Arg364, Asp533, Gln633 and 5 -thio-20 deoxyguanosine phosphonic acid of
the DNA backbone.
Figure 9. 4-arylamino-3-nitrocoumarin derivative 20.

Figure
Figure9.9.4-arylamino-3-nitrocoumarin
4-arylamino-3-nitrocoumarinderivative
derivative20.
20.
Herrera et al. synthesized a series of 3- and 7-styrylcoumarins, some of which showed anti-
Herreraetetal.al.synthesized
Herrera synthesized a series
a series 3-ofand
of colon 3- and 7-styrylcoumarins,
7-styrylcoumarins, some of which showed
proliferative activity on SW480 human adenocarcinoma cellssome of which
[48]. Among showed
them, anti-
7-(4-hydroxy-
anti-proliferative
proliferative activity activity
on on human
SW480 SW480colonhuman colon adenocarcinoma
adenocarcinoma cells [48]. cells [48].
Among them, Among them,
7-(4-hydroxy-
3,5-dimethoxystyryl)-2H-chromen-2-one (21, Figure 10) showed the highest activity (IC50 = 1.01 μM)
7-(4-hydroxy-3,5-dimethoxystyryl)-2H-chromen-2-one
3,5-dimethoxystyryl)-2H-chromen-2-one (21, (21, Figure 10) showed the(IC
highest activity
as it was capable of inducing apoptosis in Figure
SW48010) showed
cells, the by
probably highest activity
modulating 50 = 1.01 μM)
the tumor-suppressor
(IC
as it 50
was= 1.01 µM)
capable as it was capable of inducing apoptosis in SW480 cells, probably by modulating the
protein p53. of
The inducing apoptosiswas
new compound in SW480 cells,inprobably
also tested by modulating
vivo, demonstrating to the tumor-suppressor
be able to inhibit the early
tumor-suppressor
protein p53. The new protein p53. The
compound wasnew
alsocompound
tested was also tested in vivo, demonstrating to be able
progression of colon adenocarcinoma [49]. in vivo, demonstrating to be able to inhibit the early
to inhibit the
progression ofearly
colonprogression of colon
adenocarcinoma adenocarcinoma [49].
[49].
Figure 10. Chemical structure of styrylcoumarin 21.
As coumarin nucleus can be widely

Figure 10. decorated, it can beofused
Chemical structure in the synthesis
styrylcoumarin 21. of hybrid compounds,
targeting different proteinsFigure involved10. Chemical
not onlystructure
in tumorofgrowth
styrylcoumarin
but also 21.
in metastatic and angiogenetic
processes. AsIncoumarin nucleus
this context, Diao can
and be widely decorated,
collaborators synthesized it can be used
a series in the synthesis
of diethylene of hybrid
glycol tethered
As coumarin nucleus
compounds, targeting different can be widely
proteins decorated,
involved it can be used in the synthesis of hybrid
isatin-1,2,3-triazole-coumarin derivatives (22a-l, Figurenot
11) only in tumor
[50]. Several growth butoralso
isatin-based 1,2,3-intriazole
metastatic
compounds, targetingprocesses.
and angiogenetic different proteins involved
In this context, Diaonot only
and in tumor growth
collaborators but also in metastatic
or coumarin-based compounds (semaxanib, carboxyamidotriazole and synthesized a series
STX64) are involved of
in diethylene
clinical
and angiogenetic
glycol tethered processes. In this context, Diao and
isatin-1,2,3-triazole-coumarin collaborators
derivatives (22a-l, synthesized
Figure 11) a[50].series of diethylene
Several isatin-based
trials or have already been used the treatment of various cancers (as colon-rectal, prostatic, endometrial
glycol tethered isatin-1,2,3-triazole-coumarin derivatives (22a-l, Figure 11) [50]. Several isatin-based
andor 1,2,3-cancer)
breast triazole or coumarin-based
[51,52], compounds (semaxanib, derivatives
whereas isatin-1,2,3-triazole-coumarin carboxyamidotriazole
showed activityand STX64)
against are
or 1,2,3- triazole
involved or coumarin-based
in clinical or havecompounds been(semaxanib, carboxyamidotriazole and (as
STX64) are
different cancer types. trials
In addition, already
SAR studies used the treatment
demonstrated that of various
the linker cancers
between colon-rectal,
isatin and
involved in clinical
prostatic, influencestrials
endometrial or have already been used the treatment of various cancers (as colon-rectal,
1,2,3-triazole theand breast
activity cancer)
[52,53] and[51,52], whereas
that hydrogen isatin-1,2,3-triazole-coumarin
bonds are fundamental for the biological derivatives
prostatic,
showed endometrial and breast cancer) [51,52], whereas isatin-1,2,3-triazole-coumarin derivatives
activity [54].activity
These against
evidences different
guidedcancer
Diao types. In addition,
and collaborators SAR
in thestudies
choice demonstrated that theaslinker
of diethylene glycol a
showed activity
between against
isatin and different cancer influences
1,2,3-triazole types. In addition,
the SAR studies
activity [52,53] demonstrated
and that that the bonds
hydrogen linker are
linker [47]. Ten compounds were tested on HepG2 (liver carcinoma), HeLa (cervical cancer), A549 (lung
between isatin and
fundamental for 1,2,3-triazole
the biological influences theThese
activity [52,53]guided
and thatDiaohydrogen bonds are
adenocarcinoma), DU145 (prostaticactivity
cancer),[54].
SKOV3 evidences
(ovarian carcinoma), MCF-7 and collaborators
(breast cancer) and in the
fundamental for the
choice of diethylene biological
glycol activity [54]. These
as a linker [47]. TenMCF-7)evidences
compounds guided
were Diao
tested and collaborators in the
drug-resistant MCF-7/DOX (doxorubicin-resistant human cancer cell on HepG2
lines. (liver
All the carcinoma),
synthesized
choice of diethylene
HeLa (cervical glycol asA549
a linker [47].adenocarcinoma),
Ten compounds were tested on HepG2 (liver carcinoma),
compounds showedcancer),
weak to moderate (lung in vitro activity against DU145
all the(prostatic
cell lines,cancer),
thereforeSKOV3
further(ovarian
SAR
HeLacarcinoma),
(cervical cancer),
MCF-7 A549 (lung adenocarcinoma), DU145 (prostatic cancer), SKOV3 (ovarian
studies are necessary for(breast cancer) and
the obtainment drug-resistant
of new, more activeMCF-7/DOX
compounds.(doxorubicin-resistant MCF-7)
carcinoma), MCF-7 (breast cancer) and drug-resistant MCF-7/DOX (doxorubicin-resistant MCF-7)
human cancer cell lines. All the synthesized compounds showed weak to moderate in vitro activity
human cancer cell lines. All the synthesized compounds showed weak to moderate in vitro activity
against all the cell lines, therefore further SAR studies are necessary for the obtainment of new, more
against
Int. J. Mol. all
Sci. the
2020,cell lines, therefore further SAR studies are necessary for the obtainment of new,9 more
21, 4618 of 81
active compounds.
active compounds.
Figure 11. General structure of diethylene glycol tethered isatin-1,2,3-triazole-coumarin derivatives

Figure 11.General
Figure11. Generalstructure
structureofofdiethylene
diethyleneglycol
glycoltethered
tetheredisatin-1,2,3-triazole-coumarin
isatin-1,2,3-triazole-coumarinderivatives
derivatives
22a-l.
22a-l.
22a-l.
Cai
Cai et al. developed aa series
seriesofoffluorescent
fluorescentcoumarin-benzo[b]thiophene
coumarin-benzo[b]thiophene 1, 1-dioxide conjugates
Caietetal.al.developed
developed a series of fluorescent coumarin-benzo[b]thiophene 1, 1-dioxide conjugates
1, 1-dioxide [55].
conjugates
[55].
TheseThese compounds
compounds act
act on on STAT3, which is involved in the regulation of mitochondrial apoptotic
[55]. These compounds actSTAT3, which
on STAT3, is involved
which is involved in the regulation
in the regulationofof
mitochondrial
mitochondrialapoptotic
apoptotic
pathway
pathway [56]. In particular, they hypothesized that the inhibition of phosphorylation of Tyr705 and
pathway [56]. In particular, they hypothesized that the inhibition of phosphorylationofofTyr705
[56]. In particular, they hypothesized that the inhibition of phosphorylation Tyr705and and
Ser727
Ser727 might
might prevent
prevent STAT3
STAT3 activation.
activation. Four
Four STAT3
STAT3 over-activated
over-activated human
human cancer
cancer cell
cell lines
lines (i.e.,
(i.e.,
Ser727 might prevent STAT3 activation. Four STAT3 over-activated human cancer cell lines (i.e.,
human
human breast carcinoma MDA-MB-231 and MCF-7 cells, human colonic carcinoma HCT-116 cells
humanbreast
breastcarcinoma
carcinomaMDA-MB-231
MDA-MB-231and andMCF-7
MCF-7cells,cells,human
humancolonic
coloniccarcinoma
carcinomaHCT-116
HCT-116cells cells
and
and human hepatocellular carcinoma HepG2 cells) were selected to assess the biological activity of
andhuman
humanhepatocellular
hepatocellularcarcinoma
carcinomaHepG2HepG2cells)cells)were
wereselected
selectedtotoassess
assessthethebiological
biologicalactivity
activityofof
the
the newly-synthesized compounds. Compound 23 (Figure 12) showed high potency in inducing
thenewly-synthesized
newly-synthesizedcompounds. compounds. Compound
Compound 23 23 (Figure
(Figure 12)12)showed
showedhigh
highpotency
potencyinininducing
inducing
cancer
cancer cell apoptosis and ROS generation, inhibiting STAT3 phosphorylation on Tyr705, affecting
cancer cell apoptosis and ROS generation, inhibiting STAT3 phosphorylation onTyr705,
cell apoptosis and ROS generation, inhibiting STAT3 phosphorylation on Tyr705,affecting
affecting
mitochondrial
mitochondrial membrane potential and preventing STAT3 DNA-binding activity. In addition, 23
mitochondrialmembrane membranepotential
potentialandandpreventing
preventingSTAT3 STAT3DNA-binding
DNA-bindingactivity.
activity.InInaddition,
addition,23 23
inhibited
inhibited implanted
implanted 4T1
4T1 breast
breast cancer
cancer growth
growth in
in vivo.
vivo. The
The antiproliferative
antiproliferative effects
effects ofofcompound
compound 23
23
inhibited implanted 4T1 breast cancer growth in vivo. The antiproliferative effects of compound 23
on
on normal cells were investigated by MTT assays comparing it to the STAT3 inhibitor Stattic, which
onnormal
normalcellscellswere
wereinvestigated
investigatedby byMTT
MTTassays
assayscomparing
comparingitittotothe theSTAT3
STAT3inhibitor
inhibitorStattic,
Stattic,which
which
showed
showed growth
growth inhibition
inhibition activity
activity ininboth
both tumor
tumor and
and normal
normal cells,
cells, whereas
whereas compound
compound 23
23 exhibited
exhibited
showed growth inhibition activity in both tumor and normal cells, whereas compound 23 exhibited
selective
selective inhibitory activity against cancer cells (IC 50 14.62 μM for MCF-10A cells and IC50 35.60 μM
selectiveinhibitory
inhibitoryactivity
activityagainst
againstcancer
cancercells
cells(IC
(IC505014.62
14.62µMμMfor forMCF-10A
MCF-10Acellscellsand
andICIC505035.60
35.60µMμM
for
forL02
L02 cells),
cells), indicating
indicating aahigher
higher selectivity
selectivity than
than Stattic.
Stattic.
for L02 cells), indicating a higher selectivity than Stattic.
Figure 12. Chemical structure of compound 23.

2.3. Carbonic Anhydrase Inhibition
2.3. Carbonic Anhydrase Inhibition
2.3. Carbonic anhydrasesInhibition
Carbonic Anhydrase (CAs) are ubiquitous metalloenzymes that catalyze the reversible hydration
Carbonic
of carbon anhydrases
dioxide (CAs)and
to bicarbonate areprotons,
ubiquitous
whichmetalloenzymes that catalyze
is an essential physiological the reversible
reaction [57]. Thus,
Carbonic anhydrases (CAs) are ubiquitous metalloenzymes that catalyze the reversible
hydration
this enzymeof carbon dioxide
is involved in ato bicarbonate
wide range of and protons, which
physiological is an essential
and pathological physiological
processes reaction
(pH regulation,
hydration of carbon dioxide to bicarbonate and protons, which is an essential physiological reaction
[57].
CO2Thus, this enzyme
homeostasis, is involved
respiration, bonein a wide range
resorption of physiological
and tumorigenesis) and
[58] pathological
and processes
its deregulation (pH
by means
[57]. Thus, this enzyme is involved in a wide range of physiological and pathological processes (pH
regulation,
of carbonic CO 2 homeostasis,
anhydrases inhibitorsrespiration,
(CAIs) maybone resorption
be useful and tumorigenesis)
in the treatment [58] and
of many disorders its
[59–61].
regulation, CO2 homeostasis, respiration, bone resorption and tumorigenesis) [58] and its
deregulation
The ideal CAby means would
inhibitor of carbonic anhydrases
selectively inhibitors
act against those (CAIs)
isoformsmay
(hCA be IX,
useful
XII, in
forthe treatment
instance) of
related
deregulation by means of carbonic anhydrases inhibitors (CAIs) may be useful in the treatment of
many disorders
to a certain [59–61].
disease TheIn
[62,63]. ideal
thisCA inhibitor
context, would emerged
coumarins selectively
asact againstatypical
potential those isoforms (hCAthat,
hCA ligands IX,
many disorders [59–61]. The ideal CA inhibitor would selectively act against those isoforms (hCA IX,
XII, for classical
unlike instance)hCAIs,
relateddotonot
a certain
need todisease
chelate[62,63]. In this zinc
the prosthetic context, coumarins
ion but, emerged
after binding the as potential
catalytic site,
XII, for instance) related to a certain disease [62,63]. In this context, coumarins emerged as potential
are hydrolyzed to the corresponding 2-hydroxy cinnamic acid derivatives, the actual inhibitors [64,65].
Some studies highlighted that coumarins are capable of binding at the entrance of hCA catalytic site,
atypical hCA ligands that, unlike classical hCAIs, do not need to chelate the prosthetic zinc ion but,
atypical
after binding hCAthe ligands that,site,
catalytic unlike
areclassical hCAIs,todo
hydrolyzed notcorresponding
the need to chelate2-hydroxy
the prosthetic zinc ion
cinnamic acidbut,
after binding
derivatives, the catalytic
the actual inhibitors site,[64,65].
are hydrolyzed
Some studies to the corresponding
highlighted 2-hydroxy
that coumarins arecinnamic
capable ofacid
derivatives,
binding at Sci.
Int. J. Mol. thethe actual
entrance
2020,
inhibitors [64,65]. Some studies highlighted that
21, 4618 of hCA catalytic site, blocking the enzyme activity. Furthermore,10 7-
coumarins are capable of
of 81
hydroxycoumarins derivatives showed good selectivity toward IX and XII isoforms over hCA I and 7-
binding at the entrance of hCA catalytic site, blocking the enzyme activity. Furthermore,
hydroxycoumarins
II and, in some cases, they derivatives
exhibitedshowed
cytotoxicgood selectivity
activity toward
on cancer IX[66–69].
cells and XIIMany
isoforms over
efforts hCA
have I and
been
blocking
II and, in the
someenzyme
cases, activity.
they Furthermore,
exhibited cytotoxic 7-hydroxycoumarins
activity on cancer derivatives
cells
made in this direction and the most recent results are here reported. Fois and collaborators have [66–69]. showed
Many good
efforts selectivity
have been
toward
made in
recently IX and XII
this direction
investigated isoforms
the and over
the most
selective hCA I and II and,
recent activity
inhibitory in
results are some cases,
here reported.
towards they
CA IX and exhibited
FoisCA andXII cytotoxic
collaboratorsactivity
of extracts have
of
on cancer cells [66–69]. Many efforts have been made in this direction
Magydaris pastiacea seeds, from which they isolated ten linear furocoumarins, four simple coumarins of
recently investigated the selective inhibitory activity towards CA IX and
and the
CA most
XII recent
of results
extracts
andare here angular
Magydaris
a new reported.
pastiacea Fois
seeds,and collaborators
from
dihydrofurocoumarin which they [70]have recently
isolated
All the investigated
ten linear
mentioned the selective
furocoumarins,
compounds were four inhibitory
simple
tested activity
coumarins
against four
towards
and a new
isoforms CA IX and
angular
of hCA: both CA XII of extracts
dihydrofurocoumarin of
extracts showed selective Magydaris
[70] All pastiacea
the mentioned
activity seeds, from
towards compounds which
CA IX and XII they
were isolated ten
tested against
whereas linear
four
no effect
furocoumarins,
isoforms of hCA: four simple
both coumarins
extracts showed and a new
selective angular
activity dihydrofurocoumarin
towards
was seen on isoforms I and II at a concentration of 100 ng/mL. The most promising compound was CA IX and [70]
XII All the
whereas mentioned
no effect
compounds
was seen onwere
umbelliprenin (24, testedI and
isoforms
Figure against
13), II atfour
highly isoforms
active (Ki of
a concentration hCA: both
of nM)
= 5.8 100 extracts
ng/mL.
against CAshowed
The most
XII and selective
promising activity
compound
highly selective towards
overwas
CA IX and
I andXII
umbelliprenin
isoforms II. whereas
(24, Figure no 13),
effect was seen
highly activeon(Kisoforms
i = 5.8 nM)I and II at aCA
against concentration
XII and highlyof 100 ng/mL. over
selective The
most
isoforms I and II.compound was umbelliprenin (24, Figure 13), highly active (Ki = 5.8 nM) against CA
promising
XII and highly selective over isoforms I and II.
Figure 13. Chemical structure of umbelliprenin (24).

Figure
Figure 13. Chemical structure
13. Chemical structure of
of umbelliprenin
umbelliprenin (24).
(24).
Umbelliprenin (24) was then tested on HeLa cancer cells in order to evaluate its cytotoxic activity
Umbelliprenin (24) was then tested on HeLa cancer cells in order to evaluate its cytotoxic activity
Umbelliprenin
and resulted to possess (24) was then tested
a moderate on HeLa
cytotoxicity (ICcancer
50 =75 cells
μM)inproving
order totoevaluate
be ableits tocytotoxic activity
inhibit tumor
and resulted to possess a moderate cytotoxicity (IC50 = 75 µM) proving to be able to inhibit tumor
and resulted
growth, to possess
angiogenesis a moderateformation
and metastasis cytotoxicity in mice =75 μM)
(IC50 (after i.p. proving to be able
administration). to noteworthy
It is inhibit tumor
growth, angiogenesis and metastasis formation in mice (after i.p. administration). It is noteworthy
growth, angiogenesis and metastasis formation in mice (after
that CA IX and XII are overexpressed in tumor cells under hypoxic conditions, whereas i.p. administration). It is noteworthy
the
that CA IX and XII are overexpressed in tumor cells under hypoxic conditions, whereas the mentioned
that CA tests
mentioned IX and wereXIIcarried
are overexpressed in tumor
out under normoxic cells under
conditions, which hypoxic
could conditions,
explain the whereas
moderatethe
tests were carried out under normoxic conditions, which could explain the moderate cytotoxic activity
mentioned
cytotoxic testsofwere
activity carriedcompound.
the isolated out under normoxic conditions, which could explain the moderate
of the isolated compound.
cytotoxic activity of the isolated compound.
In 2019, starting from 4-methylumbelliferone (25, Figure 14), Buran and co-workers synthesized
In 2019, starting from 4-methylumbelliferone (25, Figure 14), Buran and co-workers synthesized
a seriesInof2019, starting from
8-substituted 4-methylumbelliferone
coumarin-based compounds (25,bearing
Figure 14), Buran and co-workers
alkylpiperazine synthesized
and arylpiperazine
a series of 8-substituted coumarin-based compounds bearing alkylpiperazine and arylpiperazine
a series of 8-substituted coumarin-based compounds bearing alkylpiperazine
chains (26-37, Figure 14), and evaluated their inhibitory activity against hCA I, II, IX and XII [71]. All and arylpiperazine
chains (26–37, Figure 14), and evaluated their inhibitory activity against hCA I, II, IX and XII [71].
thechains
tested(26-37,
compounds Figurewere
14), and
ableevaluated their isoforms
to inhibit hCA inhibitoryIXactivity
and XII, against hCA
without I, II, IXany
showing andinhibitory
XII [71]. All
All the tested compounds were able to inhibit hCA isoforms IX and XII, without showing any
the tested
activity towardscompounds wereisoforms
the cytosolic able to inhibit
I and IIhCA
up to isoforms
a 10 μM IXconcentration.
and XII, without Theshowing
test pointedany out
inhibitory
that
inhibitory activity towards the cytosolic isoforms I and II up to a 10 µM concentration. The test
activity towards the cytosolic isoforms I and II up to a 10 μM concentration.
these compounds had higher affinity for hCA XII over IX and, except for compound 36 (Ki (hCA XII) The test pointed out that
pointed out that these compounds had higher affinity for hCA XII over IX and, except for compound 36
these
= 26.4 compounds
nM), they all had hadKhigher affinity
i values for hCA
comparable to XII
thoseoverof IX
theand, exceptdrug
reference for compound
acetazolamide 36 (K(K
i (hCA
i (hCA XII)
(Ki (hCA XII) = 26.4 nM), they all had Ki values comparable to those of the reference drug acetazolamide
= 26.4
XII) nM).they all had Ki values comparable to those of the reference drug acetazolamide (Ki (hCA
= 5.7nM),
(Ki (hCA XII) = 5.7 nM).
XII) = 5.7 nM).
26: 33:
HO
26: O O 33: F
27: HO O O 34: F
CH3
27: 34: Br
28: CHCH
CH3
2 3
Br
28: CH 2CH3 35:
29: CH 2CH 2 OH
35:
HO O O 29: CH 2CH 2 OH
HO O O 30: CH 2CHCH 2
HO O O
HO O O N 26-37 30: CH 2CHCH 2 36:
25 F
N N 26-37 31: 36:
25 R F
N 31:
R O
32: 37:
37: O O
32: Cl
O
Cl
Figure
Figure 14.14. Chemical
Chemical structure
structure of compound
of compound 25 25
andand piperazine
piperazine derivatives
derivatives 26–37.
26–37.
Figure 14. Chemical structure of compound 25 and piperazine derivatives 26–37.
It remarkable
It is is remarkable
thatthat the substitution
the substitution of C8ofposition
C8 position of 4-methylumbelliferone
of 4-methylumbelliferone (25) did(25)
notdid
havenot
haveItany influence on
is remarkable thatinhibition of hCAofXII,
the substitution suggesting
C8 position that no significant interaction
of 4-methylumbelliferone may
(25) did not be
have
achieved between the side chains of compounds 26–37 and the catalytic site of isoform XII. On the
other hand, alkylpiperazine derivatives showed better activity on hCA IX when compared with the
other synthesized compounds, being compound 30 the one with the highest activity among them
any influence on inhibition of hCA XII, suggesting that no significant interaction may be achieved
between the side chains of compounds 26–37 and the catalytic site of isoform XII. On the other hand,
alkylpiperazine
Int. derivatives
J. Mol. Sci. 2020, 21, 4618 showed better activity on hCA IX when compared with the 11other of 81
synthesized compounds, being compound 30 the one with the highest activity among them (IC50 =
27.1 nM). Similar results were obtained by many other groups that have recently synthesized
(IC 50 = 27.1 nM).compounds
coumarin-based Similar results andwere obtained
evaluated thembyasmany
hCAother
IX andgroups that haveSulphocumarins,
XII inhibitors. recently synthesized bis-
coumarin-based compounds and evaluated them as hCA
coumarins and coumarins 1,3,4-oxadiazole derivatives are some examples [72–74]. Sulphocumarins,
IX and XII inhibitors.
bis-coumarins and coumarins 1,3,4-oxadiazole derivatives are some examples [72–74].
2.4. Antibacterial Activity
2.4. Antibacterial Activity
The battle against infections and multidrug resistant bacteria is almost certainly one of the most
The
challenging battle
issueagainst
that theinfections
scientific and multidrug
community andresistant
the whole bacteria
mankind is almost certainly
will face one future.
in the near of the
most
Multidrugchallenging
resistantissue
(MDR) that the scientific
bacteria community
are defined and the whole
as non-susceptible strainsmankind
to one orwill
more face in the near
antimicrobials
future. Multidrug resistant (MDR) bacteria are defined as non-susceptible
on three or more antimicrobial classes, whereas strains that are non-susceptible to all antimicrobialsstrains to one or moreare
antimicrobials on three or more antimicrobial classes, whereas strains that are
classified as extremely drug-resistant strains [75,76]. The plant kingdom provides a virtually endless non-susceptible to all
antimicrobials are classified as extremely drug-resistant strains [75,76]. The
source of novel chemicals and scaffolds, such as polyphenols and coumarins [77]. In 2005, the plant kingdom provides a
virtually endless source of novel chemicals and scaffolds, such as polyphenols
antibacterial potency of nearly 50 coumarin derivatives, natural and synthetic, was evaluated and then and coumarins [77].
In 2005, the
correlated byantibacterial
a SAR study.potencyBacterial ofsusceptibility
nearly 50 coumarin derivatives,
to coumarins natural and
was evaluated synthetic, was
by determining the
evaluated and then correlated by a SAR study. Bacterial susceptibility
minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), to coumarins wasconsidering
evaluated
by determining
active compounds the minimal
exhibiting inhibitory concentration
MIC values ranging from(MIC)62.5and minimum
to 2000 μg/mL. bactericidal
Among concentration
the active
(MBC),
compounds, considering
osthenoleactive
(a compounds exhibiting
natural coumarin havingMICalso
values
the ranging from 62.5 toactivity)
anti-inflammatory 2000 µg/mL.showedAmongthe
the active compounds, osthenole (a natural coumarin having
most potent activity with a MIC of 62.5 μg/mL against S. aureus and B. cereus [78]. also the anti-inflammatory activity)
showed the most
In 2015, potent activity
Nagamallu with a MIC
and co-workers of 62.5 the
exploited µg/mL against S. aureus
Vilsmeier-Haack and B.tocereus
reaction obtain[78].
a series of
In 2015, Nagamallu and co-workers exploited the Vilsmeier-Haack
novel pyrazole-containing coumarins and then evaluated their antioxidant and antibacterial reaction to obtainactivities
a series
of novel pyrazole-containing coumarins and then evaluated their
[79]. Among the series, two compounds (38 and 39, Figure 15) showed a good antibacterial andantioxidant and antibacterial
activities
antifungal[79]. Among
activity, theMIC
with series, two compounds
values comparable(38 withandthe Figure
39, ones of15) showed a good
ciprofloxacin antibacterial
(positive control
and antifungal activity, with MIC values comparable with the ones
against bacteria species) and fluconazole (positive control against fungi strains). of ciprofloxacin (positive control
against bacteria species) and fluconazole (positive control against fungi strains).
Figure 15. Chemical structure and minimal inhibitory concentration (MIC) values of antibacterial
Figure 15. Chemical structure and minimal inhibitory concentration (MIC) values of antibacterial
coumarins proposed by Nagamallu and colleagues [79].
coumarins proposed by Nagamallu and colleagues [79].
Following the idea that the introduction of an additional coumarin nucleus on a parent coumarin
Following the idea that the introduction of an additional coumarin nucleus on a parent coumarin
molecule could improve the pharmacological activity (i.e., dicumarol as anticoagulant), in 2017,
molecule could improve the pharmacological activity (i.e., dicumarol as anticoagulant), in 2017,
Chougala and colleagues designed and synthesized a series of bis-coumarin derivatives [80].
Chougala and colleagues designed and synthesized a series of bis-coumarin derivatives [80].
Figure 16 shows the common scaffolds of the bis-coumarins synthesized using l-proline as catalyst
Figure 16 shows the common scaffolds of the bis-coumarins synthesized using L-proline as
in a multi-component reaction approach.
catalyst in a multi-component reaction approach.
Int. J. Mol. Sci. 2020, 21, 4618 12 of 81
Figure 16. Coumarin dimer scaffolds proposed by Chougala and colleagues and the corresponding
Figure 16.
16. Coumarin dimer
dimer scaffolds
scaffolds proposed
proposed by
by Chougala
Chougala and
and colleagues
colleagues and
and the
thecorresponding
corresponding
MIC valuesCoumarin
Figure [80].
MIC values [80].
MIC values [80].
The antibacterial potency of the compounds was evaluated against Gram-positive and Gram-
The antibacterial
The antibacterial potency
potencyofofthethe compounds
compounds waswas evaluated
evaluatedagainst Gram-positive
against Gram-positiveand Gram-
and
negative strains, comparing the MIC with ciprofloxacin and most of the newly synthesized
negative strains,
Gram-negative comparing
strains, comparing the MIC
the MICwithwith ciprofloxacin
ciprofloxacin andand most
mostofof the
the newly synthesized
synthesized
compounds showed modest to good inhibiting activity against the tested microorganisms.
compoundsshowed
compounds showed modest
modest to good
to good inhibiting
inhibiting activitythe
activity against against the tested microorganisms.
tested microorganisms. Compounds
Compounds 40a–e (Figure 16) were highly active and more potent than ciprofloxacin against S. aureus
40a–e (Figure 16) were highly active and more potent than ciprofloxacin against S. aureus and E.S.faecalis,
Compounds 40a–e (Figure 16) were highly active and more potent than ciprofloxacin against aureus
and E. faecalis, whereas 41c and 41d were active only against Gram-positive E. faecalis. Actually,
and E. faecalis,
whereas 41c andwhereas
41d were41c and only
active 41d against
were active only against
Gram-positive Gram-positive
E. faecalis. Actually,E.compounds
faecalis. Actually,
41a–e
compounds 41a–e were the most promising against E. coli.
compounds 41a–e were the most
were the most promising against E. coli. promising against E. coli.
Once established the broad spectrum of action of the coumarin nucleus, various researchers
Once established
Once established the the broad
broad spectrum
spectrum of of action
action ofof the
the coumarin
coumarin nucleus,
nucleus, various
various researchers
researchers
focused their attention on the activity against multidrug resistant strains, in particular on the ESKAPE
focusedtheir
focused their attention
attentionon on the
the activity
activity against
against multidrug
multidrug resistant
resistantstrains,
strains,inin particular
particularon on the
the ESKAPE
ESKAPE
pathogens, the coterie that escape the lethal action of antibiotics: Enterococcus faecium, Staphylococcus
pathogens, the coterie that escape the lethal action of antibiotics: Enterococcus
pathogens, the coterie that escape the lethal action of antibiotics: Enterococcus faecium, Staphylococcus faecium, Staphylococcus
aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa and Enterobacter species
aureus,Klebsiella
aureus, Klebsiellapneumoniae,
pneumoniae,Acinetobacter
Acinetobacter baumanii,
baumanii, Pseudomonas
Pseudomonas aeruginosa
aeruginosa and and Enterobacter
Enterobacter species
species [81].
[81]. In 2017, Naik et al. synthesized and evaluated against S. aureus and other bacterial strains a series
[81].
In In 2017,
2017, NaikNaik
et al.etsynthesized
al. synthesized and and evaluated
evaluated against
against S. aureus
S. aureus andand other
other bacterial
bacterial strains
strains a series
a series of
of 3,4-dihydropyrimidinone-coumarin analogues, measuring the MIC values and comparing them to
of 3,4-dihydropyrimidinone-coumarin analogues, measuring the MIC
3,4-dihydropyrimidinone-coumarin analogues, measuring the MIC values and comparing them to values and comparing them to
ciprofloxacin [82].
ciprofloxacin[82].
ciprofloxacin [82].
The structures and the MIC values for compounds 42–49, the most promising derivatives, are
The structures
The structures and and thethe MIC
MIC values
values for
for compounds
compounds 42–49, 42–49, thethe most
most promising
promising derivatives,
derivatives, are are
reported in Figure 17: the substitution at C6 position seemed to be excellent for the activity and able
reported in Figure 17: the substitution at C6 position seemed to be excellent
reported in Figure 17: the substitution at C6 position seemed to be excellent for the activity and able for the activity and able
to modulate the potency, decreasing the efficiency in the order of -CH3 > 7,8-Benzo > -Cl > -OCH3.
3 > 7,8-Benzo
to modulate
to modulate thethe potency,
potency,decreasing
decreasingthe theefficiency
efficiencyininthe the orderofof-CH -CH 3 > 7,8-Benzo> > -Cl >> -OCH
-OCH3.
Furthermore, it was revealed from docking studies that order
that one hydrogen atom and-Cl two oxygen3 .
Furthermore, it was revealed from docking studies that that one hydrogen
Furthermore, it was revealed from docking studies that that one hydrogen atom and two oxygen atoms atom and two oxygen
atoms of 3,4-dihydropyrimidinone substituted coumarins form interactions with the active site
atoms
of of 3,4-dihydropyrimidinone
3,4-dihydropyrimidinone substituted coumarins form interactions withsitethe active ofsite
S.
residues of S. aureus gyrase,substituted
indicatingcoumarins form interactions
that the presence of hydrogen with the active
bond acceptor residues
and donor
residues
aureus of S.indicating
gyrase, aureus gyrase,
that theindicating
presence that
of the presence
hydrogen bond of hydrogen
acceptor and bond groups
donor acceptor may and donor
enhance
groups may enhance antimicrobial activity.
groups may enhance
antimicrobial activity. antimicrobial activity.
H H Minimum inhibitory concentration

O NH S NH Minimum
(MIC) inhibitory
values concentration
in in g/mL - S.aureus
O N S N (MIC) values in in g/mL - S.aureus
HN O HN O
HN O HN O
42 0.4
R1 O R2 O 42
43 0.4
0.8
R1 O R2 O 43 0.8
44 0.2
44
45 0.2
0.4
O O O O
O O O O 45
46 0.4
0.4
46
47 0.4
0.8
42 R1 =6-Me 46 R2 =6-Me
42 46 47 0.8
43 =6-Me
RR11 =6-OMe 47 =6-Me
RR22 =6-OMe 48 0.8
0.8
43 R =6-OMe 47 R =6-OMe 48
49 0.4
44 R1 =7-Me
1 48 R2 =6-Cl
2
0.4
44 48 49
45 =7-Me
RR11 =7,8-Benzo 49 =6-Cl
RR22 =7,8-Benzo Ciprofloxacin 2
2
45 R1 =7,8-Benzo 49 R2 =7,8-Benzo Ciprofloxacin
Figure 17. 3,4-dihydropyrimidinone-coumarin analogues and their MIC values against S. aureus.
In 2018, Chavan and Hosamani proposed a facile method for the microwave assisted synthesis
of a series
In 2018,ofChavan
pyrazole-containing
and Hosamani coumarins
proposedand tested
a facile their for
method antibacterial and anti-inflammatory
the microwave assisted synthesis
In 2018, Chavan and Hosamani proposed a facile method for the microwave assisted synthesis
activities
of a series[83]. The researchers evaluated
of pyrazole-containing the inand
coumarins vitro antibacterial
tested activity of and
their antibacterial the newly synthesized
anti-inflammatory
of a series of pyrazole-containing coumarins and tested their antibacterial and anti-inflammatory
activities
compounds [83]. The researchers
through evaluated
agar-well diffusion the in against
method vitro antibacterial activity (Bacillus
two Gram-positive subtilis
of the newly synthesized
(ATCC no.
activities [83]. The researchers evaluated the in vitro antibacterial activity of the newly synthesized
Int.J. J.Mol.
Int. Mol.Sci.
Sci.2020,
2020,21,
21,x xFOR
FORPEER
PEERREVIEW
REVIEW 1313ofof8383
compounds
compounds
Int. through
J. Mol. Sci.through
2020, agar-welldiffusion
agar-well
21, 4618 diffusionmethod
methodagainst
againsttwo
twoGram-positive
Gram-positive(Bacillus
(Bacillussubtilis
subtilis(ATCC
(ATCC
13 of 81
no.23857)
no. 23857)and andStaphylococcus
Staphylococcusaureus
aureus(ATCC-29213))
(ATCC-29213))and andtwotwoGram-negative
Gram-negative(Escherichia
(Escherichiacoli
coli(ATCC-
(ATCC-
25922)and
25922) andPseudomonas
Pseudomonasaeruginosa
aeruginosa(ATCC
(ATCCNo.25619))
No.25619))bacterial
bacterialstrains
strains[84].
[84].The
TheMIC
MICvalues
valueswere
were
23857) and Staphylococcus aureus (ATCC-29213)) and two Gram-negative (Escherichia coli (ATCC-25922)
compared to those of ciprofloxacin and all the compounds showed significant
compared to those of ciprofloxacin and all the compounds showed significant antibacterial activity.antibacterial activity.
and Pseudomonas aeruginosa (ATCC No.25619)) bacterial strains [84]. The MIC values were compared
InInparticular,
particular,compounds
compounds5050and and5151(Figure
(Figure18),
18),showed
showedan anexcellent
excellentactivity
activityagainst
againstS.S.aureus
aureus(MIC
(MIC
to those of ciprofloxacin and all the compounds showed significant antibacterial activity. In particular,
0.78μg/mL
0.78 μg/mLand andMICMIC1.562
1.562μg/mL,
μg/mL,respectively).
respectively).Docking
Dockingstudies
studieswere
wereiningood
goodagreement
agreementwith withthe
the
compounds 50 and 51 (Figure 18), showed an excellent activity against S. aureus (MIC 0.78 µg/mL and
biologicalresults.
biological results.
MIC 1.562 µg/mL, respectively). Docking studies were in good agreement with the biological results.
OO
NN
NN
OO NN
RR
OO OO
5050RR= =6-Cl
6-Cl
5151RR= =7,8-Benzo
7,8-Benzo
Figure18.
Figure
Figure 18.Coumarin-pyrazole
18. Coumarin-pyrazole hybridsand
Coumarin-pyrazolehybrids
hybrids andtheir
and theirMIC
their MICvalues
MIC valuesagainst
values againstS.S.
against S.aureus.
aureus.
aureus.
In2017,
InIn 2017,Madeiro
2017, Madeiro
Madeiro and
and
and co-workers
co-workers
co-workers focalized
focalized
focalized theirinterest
theirtheir interest
interest towards
towards
towards theantibiotic
the
the antibiotic antibiotic activity
activity ofactivity
coumarinsofof
coumarins
coumarins
isolated from isolated
isolated
Rutacea from
from Rutacea
Rutacea
species species
species
(Figure (Figure
(Figure
19) [85]. 19) [85].
19) [85].
Bergapten, Bergapten,
Bergapten,
xantoxin, xantoxin, isopimpinellin
xantoxin, isopimpinellin
isopimpinellin and imperatorinand and
did
imperatorin
imperatorin
not exhibit any did
did notexhibit
not exhibitactivity,
antibacterial anyantibacterial
any antibacterial activity,
even at theactivity, evenatatthe
highest even thehighest
concentration, highest concentration,
concentration,
against S. aureus strains against
against S.S.
resistant
aureus
aureus strains
strains resistant
resistant to
to tetracycline,
tetracycline, erythromycin
erythromycin and
and norfloxacin.
norfloxacin. However,
However,
to tetracycline, erythromycin and norfloxacin. However, their role as modulator of other antibiotics their
their role
role as
as
modulator
modulator
seemed quite of other
of other antibiotics
antibiotics
promising, because seemed
seemed quite promising,
quite promising,
isopimpinellin because isopimpinellin
because reduced
and imperatorin isopimpinellin
the MICand and imperatorin
imperatorin
of erythromycin,
reducedthe
reduced theMIC
tetracycline MICof
and oferythromycin,
erythromycin,
norfloxacin. tetracycline
tetracycline
Nevertheless, moreand
and norfloxacin.
norfloxacin.
detailed Nevertheless,
Nevertheless,
research is necessary more
more detailed
detailed
in order research
research
to enable the
isnecessary
isusenecessary inin order
order toto enable
enable the
the use
use of
of these
these natural
natural products
products
of these natural products as adjuvants to antimicrobial agents. as
as adjuvants
adjuvants toto antimicrobial
antimicrobial agents.
agents.
Figure19.
Figure
Figure 19.Coumarins
19. Coumarinsisolated
Coumarins isolatedfrom
isolated fromRutacea
from Rutaceaspecies.
Rutacea species.
species.
In2018,
InIn 2018,Widelsky
2018, Widelskyand
Widelsky andco-workers
and co-workersisolated
co-workers isolatedsome
somesimilar
some similarlinear
similar linear
linear furanocoumarins
furanocoumarins
furanocoumarins from
from
from the
the
the fruits
fruits
fruits of
ofPeucedanum
Peucedanum
ofPeucedanum luxurians
luxurians
luxurians Tamamsch,
Tamamsch,
Tamamsch, with
with
with more
more
more encouraging
encouraging
encouraging results.
results.
results. Plants Plants
Plants ofofPeucedanum
of the thePeucedanum
the Peucedanum
genus genus
genus
have
havebeen
have
been beenused
used used forcenturies
for
for centuriescenturies asasantibacterial
antibacterial
as antibacterial agents
agents
agents and,
and,
and, forsome
for for
some some ofofthem,
of them, them, the
thethe activity
activity
activity was
was
was confirmed
confirmed
confirmed by
bybybiological
biological
biological and and
and pharmacological
pharmacological
pharmacological studies
studies
studies onplant
on
on plant plant extracts
extracts
extracts and on and
and ononaisolated
a few afew
fewisolated
isolated compounds
compounds
compounds [86].
[86].
[86]. All the
Allthe
All
six thesixsixisolated
isolated isolated
compounds compounds
compounds
showedshowedashowed abroad
broadgrowth-inhibitor
growth-inhibitor
broad agrowth-inhibitor activity against activity
activity against
against
several several
several
bacteria bacteria
bacteria
strains but
strains
strains
three but
but
of three
three
them ofofthem
(Figure them (Figure20)
(Figure
20) resulted 20)resulted
resultedinteresting.
particularly particularly
particularly 0 0
interesting.
6interesting.
,7 6′,7′-Dihydroxybergamottin
6′,7′-Dihydroxybergamottin
-Dihydroxybergamottin was the most
wasthe
was
active the mostactive
most
against active
all the against
against allstrains
all
bacteria thebacteria
the bacteria strains
strains
tested, tested,
tested,
probably probably
probably
because because
because
of the ofofthe
aliphatic the aliphatic
aliphatic
chain chaininin
in C5 chain
position;
C5C5 position;
position;
similar similar
similar
activity activity
wasactivity
noticedwas was noticed for peucedanin
noticed for peucedanin
for peucedanin and officinalin and officinalin
andisobutyrate. isobutyrate.
officinalin isobutyrate.
Int. J. Mol. Sci. 2020, 21, 4618 14 of 81
Figure 20. Linear coumarins isolated from the fruits of Peucedanum luxurians Tamamsch, zone of
inhibition (mm) and minimum inhibitory concentration (MIC) values.
Figure
Liu and Linear coumarins
20. colleagues isolated
madeisolated
a hugefrom the fruits of Peucedanum luxurians Tamamsch, zone of
Figure 20. Linear coumarins fromeffort to synthesize
the fruits of Peucedanumand identify
luxurians coumarin-pyrazole
Tamamsch, zone of
inhibition
carboxamide (mm) and
derivatives minimum
as inhibitory
potential concentration
topoisomerase-II (MIC) values.
inhibitors: 70 novel compounds were obtained
inhibition (mm) and minimum inhibitory concentration (MIC) values.
and evaluated [87].
Liu and colleagues made a huge effort to synthesize and identify coumarin-pyrazole carboxamide
Three
Liu and of them (52–54,made
colleagues Figurea 21) wereeffort
huge endowed with promising
to synthesize antimicrobial
and identify activities. In
coumarin-pyrazole
derivatives as potential topoisomerase-II inhibitors: 70 novel compounds were obtained and
particular,
carboxamide compound
derivatives52asshowed a topoisomerase-II
potential considerable inhibitory activity
inhibitors: compared
70 novel with were
compounds ciprofloxacin
obtained
evaluated [87].
against Escherichia
and evaluated [87]. coli and compound 53 exhibited excellent antibacterial activity against Salmonella
Three of them (52–54, Figure 21) were endowed with promising antimicrobial activities. In
typhi.Three
The selected
of them compounds exhibited
(52–54, Figure also potent
21) were endowedinhibition against Topo
with promising II and Topo activities.
antimicrobial IV with ICIn50
particular, compound 52 showed a considerable inhibitory activity compared with ciprofloxacin against
values in thecompound
particular, range 9.4–2552 mg/L.
showed a considerable inhibitory activity compared with ciprofloxacin
Escherichia coli and compound 53 exhibited excellent antibacterial activity against Salmonella typhi. The
against Escherichia coli and compound 53 exhibited excellent antibacterial activity against Salmonella
selected compounds exhibited also potent inhibition against Topo II and Topo IV with IC50 values in
typhi. The selected compounds exhibited also potent inhibition against Topo II and Topo IV with IC50
the range 9.4–25 mg/L.
values in the range 9.4–25 mg/L.
Figure 21. Selected coumarin-pyrazole carboxamide derivatives developed by Liu et al. and their MIC
values [87].
Figure 21. Selected

2.5. Antifungal Activitycoumarin-pyrazole carboxamide derivatives developed by Liu et al. and their MIC
values [87].
Fungal diseases are a well-known plague for animal and plant worlds but a more hidden menace
for human health.
2.5. Antifungal More than 90% of all reported fungal-related deaths results from species that belong
Activity
Figure 21. Selected coumarin-pyrazole carboxamide derivatives developed by Liu et al. and their MIC
to one of four genera: Cryptococcus, Candida, Aspergillus and Pneumocystis [88–90]. Coumarin derivatives
values [87].
Fungal diseases are a well-known plague for useful
animalin and plant worlds but a more hidden
are endowed with antifungal activity, potentially both pharma and agri-food sectors.menace
In this
for human
paragraph, health.
we More
will focusthan
on 90%
the of all
recent reported fungal-related
progresses in the deaths
development results
of from
novel species
antifungal that belong
drugs for
2.5. Antifungal Activity
to one of four genera: Cryptococcus, Candida, Aspergillus and Pneumocystis
human use whereas agri-food applications can be found in Section 3.2, Food systems. [88–90]. Coumarin derivatives
are endowed
Diseases with
Fungal diseases antifungal
by Candida activity,
are aspecies,
well-known potentially
a familyplague for useful
of fungi animal inand
both
that normally pharma
plant
liveworldsand
on the agri-food
but
host sectors.
a epithelial
more hidden In this
menace
species but
for human
can initiatehealth.
a fatal More thanin90%
infection of all reported
particular fungal-related
cases like deaths results
immunodeficiency, from
are the species
fourth most that belong
common
to oneof
cause of nosocomial
four genera: blood-stream
Cryptococcus, Candida, Aspergillus
infections. Despiteand Pneumocystis
several [88–90].
species of CandidaCoumarin
can cause derivatives
disease,
are endowed with antifungal activity, potentially useful in both pharma and agri-food
Candida albicans prevails in term of incidence [88,91,92]. Therefore, in 2016, Shaik and colleagues sectors. In this
paragraph, we will focus on the recent progresses in the development of novel antifungal drugs for
paragraph, we will focus on the recent progresses in the development of novel antifungal drugs for
human use whereas agri-food applications can be found in paragraph 3.2, Food systems.
Int. J. Mol.
human use Sci. 2020, 21,
whereas 4618
agri-food applications can be found in paragraph 3.2, Food systems. 15 of 81
Diseases by Candida species, a family of fungi that normally live on the host epithelial species
Diseases by Candida species, a family of fungi that normally live on the host epithelial species
but can initiate a fatal infection in particular cases like immunodeficiency, are the fourth most common
but can initiate a fatal infection in particular cases like immunodeficiency, are the fourth most common
cause of nosocomial
designed a novelblood-stream infections.
series of coumarin Despite several
derivatives species
conjugated withof 1,2,3-triazole
Candida can cause disease,
moieties, on Candida
the basis of
cause of nosocomial blood-stream infections. Despite several species of Candida can cause disease, Candida
albicans prevailswork
a previous in term byof incidence
Shi and Zhou [88,91,92]. Therefore,
and of the common in 2016,
use ofShaik and
azoles ascolleagues
antifungaldesigned a novelThe
drugs [93,94].
albicans prevails in term of incidence [88,91,92]. Therefore, in 2016, Shaik and colleagues designed a novel
series of coumarin
antifungal potencyderivatives conjugated
of the novel with 1,2,3-triazole
compounds was testedmoieties, on the basis
against Candida of aand
albicans previous
other work by
four fungal
series of coumarin derivatives conjugated with 1,2,3-triazole moieties, on the basis of a previous work by
Shi and Zhou and
pathogens (i.e.,ofFusarium
the common use of azoles
oxysporum, as antifungal
Aspergillus drugs [93,94].
flavus, Aspergillus Theand
niger antifungal potency
Cryptococcus of the
neoformans);
Shi and Zhou and of the common use of azoles as antifungal drugs [93,94]. The antifungal potency of the
novelMIC compounds
values were wasevaluated
tested against
and Candida
compared albicans
to MIC and of
other
the four fungalcompounds
reference pathogens (i.e., Fusariumand
miconazole
novel compounds was tested against Candida albicans and other four fungal pathogens (i.e., Fusarium
oxysporum, Aspergillus
fluconazole. flavus, Aspergillus
Compounds 55–57 andniger and Cryptococcus
59 (Figure neoformans);
22) were found MIC values
to be equipotent were evaluated
to miconazole against
oxysporum, Aspergillus flavus, Aspergillus niger and Cryptococcus neoformans); MIC values were evaluated
andC. compared to MIC
albicans and of the reference
compound 58 was compounds
found to be miconazole
two-fold more and active
fluconazole.
comparedCompounds 55–57 andand
with miconazole
and compared to MIC of the reference compounds miconazole and fluconazole. Compounds 55–57 and
59 (Figure 22) were
equipotent found to be equipotent to miconazole against C. albicans and compound 58 was found
to fluconazole.
59 (Figure 22) were found to be equipotent to miconazole against C. albicans and compound 58 was found
to be two-fold more active compared with miconazole and equipotent to fluconazole.
to be two-fold more active compared with miconazole O and equipotent to fluconazole.
O
O
O Minimum inhibitory concentration values
R1 N O O O Minimum
in g/mLinhibitory concentration
(MIC) against values
C. albicans
R1 O O O in g/mL (MIC) against C. albicans
N N
N
R1 N N 55 25
R1 55 R1=H R2=H R3=Cl 5655 2525
R3 5655RR 1=H
1=H RR 2=HRR
2=Cl 3=Cl
3=H 5756 2525
R3 56 R =H R =Cl
57 R1=Cl R2=H R3=H
1 2 R 3=H 5857 25
12.5
5958 12.5
25
5857RR
1 =H
1 =Cl
R R=H
2 2 =H
R R=F
3 3
=H
59 2525
5958RR
1 =H
1 =HRR=H
2 2 =HRR=H
3 3 =F Miconazole
25
59 R1=H R2=H R3=H Miconazole
Fluconazole 12.5
Fluconazole 12.5
Figure
Figure 22. Antifungal
22. Antifungal coumarins.
coumarins.
Figure 22. Antifungal coumarins.
Furthermore,
Furthermore, molecular
molecular docking
docking studies
studies showed
showed thatthat
these these compounds
compounds havehave a high
a high affinity
affinity
Furthermore,
toward the active molecular docking studies showed that these compounds have a high affinity
toward the active site site of enzyme
of enzyme P450P450 cytochrome
cytochrome lanosterol
lanosterol 14α-demethylase
14α-demethylase andand analysis
analysis of ADME
of ADME
toward the active
parameters site of enzyme
confirmed their P450 cytochrome
drug-like properties lanosterol
[94]. 14α-demethylase and analysis of ADME
parameters confirmed their drug-like properties [94].
parameters confirmed
In 2017, fifteen their drug-like
novel coumarin properties
derivatives [94].
were synthesized by Tiwari et under
al., under solvent
In 2017, fifteen novel coumarin derivatives were synthesized by Tiwari et al., solvent freefree
In 2017,
conditions fifteen novel coumarin derivatives were synthesized by Tiwari et al., under solvent free
conditions andand exploiting
exploiting the the
ionicionic
liquidliquid
[Et3[Et 3 NH][HSO
NH][HSO 4] as4 ]aas a catalyst
catalyst [95].[95].
TheThe compounds
compounds werewere
conditions and exploiting the ionic liquid [Et 3NH][HSO4] as a catalyst [95]. The compounds were
tested both for their antifungal and antibacterial activities and, among the series, compounds 60 andand
tested both for their antifungal and antibacterial activities and, among the series, compounds 60
tested both fortotheir
61 resulted antifungal
be the most andas
potent antibacterial
fungicides activities
(Figure and,
23). The among
MIC the series,
values compounds
observed 60 and 60
forcompound
compound
61 resulted to be the most potent as fungicides (Figure 23). The MIC values observed for
61 and
resulted to be the
werecomparable
61were most
comparableto potent
tothe as fungicides
thestandard
standarddrug (Figure
drugmiconazole. 23).
miconazole. The MIC values observed for compound
60 and 61
60 and 61 were comparable to the standard drug miconazole.
OH
O OH
R O
R
OH
OH
N
N
O O
O O
60 R=Me
6160R=Et
R=Me
61 R=Et
Minimum inhibitory concentration in g/mL (MIC)
Minimum inhibitory concentration in g/mL (MIC)
C. albicams C. glabrata F. oxysporum A. fumigates A. flavus A. niger C. neoformans
60 C. albicams
25 glabrata F. oxysporum
C. 30 28 A. fumigates
38 A.12
flavus A.15niger C. neoformans
15
6160 2525 2830 2828 3638 1512 1215 1215
61
Miconazole 2525 2528 2528 3536 1215 1212 1212
Miconazole 25 25 25 35 12 12 12
Figure
Figure 23. 23. Antifungal
Antifungal coumarins
coumarins synthesizedunder
synthesized undersolvent-free
solvent-free conditions
conditions and
andexploiting
exploitingionic liquid
ionic
Figure
as 23. Antifungal coumarins synthesized under solvent-free conditions and exploiting ionic
catalyst.
liquid as catalyst.
liquid as catalyst.
Further studies demonstrated that compound 60 acts by inhibiting ergosterol biosynthesis in C.
albicans. Molecular docking studies revealed, as for previous study on compounds 55–59, a highly
spontaneous binding ability of the tested compounds to the active site of lanosterol 14α-demethylase,
Further
Int. J. Mol. studies
Sci. 2020, demonstrated
21, x FOR PEER REVIEWthat compound 60 acts by inhibiting ergosterol biosynthesis 16 of in83C.
albicans. Molecular docking studies revealed, as for previous study on compounds 55–59, a highly
Further studies
spontaneous binding
Int. J. Mol. Sci. 2020,
demonstrated
ability of thethat
21, 4618
compound
tested compounds 60 acts
to the byactive
inhibiting
site ofergosterol
lanosterolbiosynthesis
14α-demethylase,in16C.of 81
albicans. Molecular
which suggests docking
that the testedstudies revealed,inhibit
compounds as for the
previous
synthesisstudy of on
thiscompounds 55–59, a in
enzyme. Moreover, highly
silico
spontaneous
ADMET properties binding ability of the tested
were evaluated andcompounds
demonstrated to thethat
activeallsite
theofcompounds
lanosterol 14α-demethylase,
exhibited a good
which
which
percent suggests
suggests
absorption that
that thethe
(% ABS) tested
tested compounds
compounds
ranging inhibit
inhibit
from 84.9% tothe the synthesis
synthesis
100%. Moreover, of of
thisthis enzyme.
enzyme.
these Moreover,
Moreover,
compounds in in
had proven silico
silico to
ADMET
ADMET
be safe after properties
properties
in vitrowerewere evaluated
toxicity, in vivo and and demonstrated
acutedemonstrated
oral toxicity andthat all the compounds
thatbehavioral
all the compounds exhibited a good
studies. exhibited a good percent
absorption
percent (% ABS)
absorption
Coumarin-based ranging
(% ABS) from
ranging
antifungal 84.9%
fromhad
azoles to 100%.
84.9%been Moreover,
to 100%.
further thesethese
Moreover,
investigated compounds
bycompounds
Elias andhadco-workers,
proven to be
had proven safe
to
who,
beinafter
safe in vitro
after in toxicity,
vitro in
toxicity, vivo in acute
vivo oral
acute toxicity
oral and
toxicity behavioral
and studies.
behavioral
2019, developed a series of 11 coumarins conjugated with 1,2,4-triazole and imidazole motifs [96]. studies.
Coumarin-based
TheCoumarin-based
analysis antifungal
antifungal
of the biological effectsazoles
azoles hadhad
of these been
been
novel further
further
chemical investigated
investigated byby Elias
Elias
entities highlighted and and
that co-workers,
co-workers, who,
imidazole-based who,
in 2019,
inderivatives
2019, developed
developed
(Figurea 24) a series
serieswere of of
11
more11 coumarins
coumarins conjugated
efficientconjugated
against with
several with 1,2,4-triazole
1,2,4-triazole
Candida strainsand and imidazole
imidazole
compared motifs
motifs [96].
to 1,2,4-triazole [96].
The The analysis
analysis of
derivatives. of the biological
the biological
Moreover, the mode effects of
effectsofofactionthese
these of novel
novel chemical
the chemical
two classes entities
entities highlighted
highlighted
of compounds that imidazole-based
thatdifferent.
were imidazole-based
Whereas
derivatives
derivatives
the antifungal (Figure
(Figure 24)24)
activity ofwere
were themore more efficient
efficient
triazole-based against
against
azoles several
several
was Candida
Candida
dependent onstrains
strains compared
compared
expression to to 1,2,4-triazole
1,2,4-triazole
of CYP51, the target
ofderivatives.
derivatives. Moreover,
the azoleMoreover,
antifungals, thethemode mode of of
imidazole-based action
action of of
thethe
two
compounds two classes
classes
displayedof of compounds
compounds
antifungal were
were different.
different.
activity a Whereas
againstWhereas
mutant
the the
lacking antifungal
antifungal
CYP51, activity
activity of the
of thethat
indicating triazole-based
triazole-based
imidazole-based azoles
azoles was was dependent
azoledependent
antifungals on
onhave expression
expression of CYP51,
of CYP51,
additional modesthe the target
of target
action.
of
thethe
ofThis azole
azole
peculiarity antifungals,
antifungals,
could be imidazole-based
imidazole-based
favorable compounds
in thecompounds
struggle displayed
displayed
against antifungal
antifungal
drugs-resistant activity
activity
fungal against
against
strains. a mutant
a mutant
lacking
lacking CYP51,
CYP51, indicating
indicating thatthat imidazole-based
imidazole-based azole
azole antifungals
antifungals havehave additional
additional modes
modes of of action.
action.
This peculiarity could be favorable in the struggle against
This peculiarity could be favorable in the struggle against drugs-resistant fungal strains.drugs-resistant fungal strains.
Figure 24. Coumarin derivatives conjugated to an imidazole-containing moiety.
Figure 24. Coumarin derivatives conjugated to an imidazole-containing moiety.

Furthermore,
Figureit24.
should
Coumarinnot be forgottenconjugated
derivatives the contribution of natural coumarins
to an imidazole-containing to the fight against
moiety.
Candida infections. Ferulago
Furthermore, it shouldtrachycarpa Boiss.the
not be forgotten is one of the species
contribution of Ferulago
of natural coumarinsW. Koch.,
to the common in
fight against
Furthermore,
Anatolian region, it should
exploited not be
in forgotten
traditional the contribution
medicine but of
alsonatural
in coumarins
culinary
Candida infections. Ferulago trachycarpa Boiss. is one of the species of Ferulago W. Koch., common in to
field. the fight
Previous against
studies
Candida
showed infections.
Anatolianthatregion, Ferulago
coumarins aretrachycarpa
exploited the Boiss.
main compounds
in traditional is onefound
medicine of
butthe species
in
also Ferulago
in of Ferulago
culinary but W.in Koch.,
onlyPrevious
field. common
2018,studies
Dikpinar in
and
showed
Anatolian
colleagues region,
conducted
that coumarins exploited
are thethemain incompounds
first traditionalfound
antimicrobial medicine
activity but also
guided
in Ferulago in
isolation
but onlyculinary
in the field.
of2018, molecularPrevious
Dikpinar studiesof
constituent
and colleagues
showed
this that
particularcoumarins
species are
of the main
Ferulago compounds
[97]. Four found
coumarin in Ferulago
compounds but only
(Figure in 2018,
25)
conducted the first antimicrobial activity guided isolation of the molecular constituent of this particular wereDikpinar
isolated and
and
colleagues
purified conducted
and then the
three first
of antimicrobial
them were activity
tested guided
against isolation
bacterial and of the molecular
fungal
species of Ferulago [97]. Four coumarin compounds (Figure 25) were isolated and purified and then strains;constituent
in of
particular,
this particular
marmesin
three of them species
senesioate, ofsuberosin
were tested Ferulago
against [97].
and Four coumarin
crenulatin
bacterial and showed
fungalcompounds
antifungal
strains; in (Figure
activity
particular,25)with
were625
marmesinisolated
mg/L and
MIC
senesioate,
purified
against and
Candidathen three
albicans. of them were tested against bacterial and fungal
suberosin and crenulatin showed antifungal activity with 625 mg/L MIC against Candida albicans. strains; in particular,
marmesin senesioate, suberosin and crenulatin showed antifungal activity with 625 mg/L MIC
against Candida albicans.
Figure25.
Figure Coumarinsfrom
25.Coumarins Ferulagotrachycarpa.
fromFerulago trachycarpa.
The antifungal
The antifungal potency
potency ofof
coumarin
Figure coumarin
25.
itselfitself
Coumarins
against Candida
fromagainst
albicansalbicans
Candida
Ferulago trachycarpa.
had been hadpreviously evaluated,
been previously
in particular its antibiofilm activity [98,99]. Recently, Xu and co-workers focused
evaluated, in particular its antibiofilm activity [98,99]. Recently, Xu and co-workers focused their their attention on
the possible
attention way
The antifungal to prevent
potency
on the possible wayofthe adhesion
to coumarin and
prevent theitself formation
adhesionagainst of biofilm
andCandida
formation by Candida
albicans albicans
had by
of biofilm been on implanted
previously
Candida albicans
medical
evaluated, devices.
in The
particular research
its group
antibiofilm observed
activity that coumarin
[98,99]. not
Recently, only
Xu suppresses
and
on implanted medical devices. The research group observed that coumarin not only suppresses co-workersbiofilm formation
focused theirbut
also affects
attention the structure
on the possible
biofilm formation but alsoof
way the mature
to prevent
affects biofilm; moreover
the adhesion
the structure the adhesion,
and formation
of the mature the cell
of biofilmthe
biofilm; moreover surface hydrophobicity
by adhesion,
Candida albicans
the cell
on (CSH)
surface and the
implanted filamentous
medical
hydrophobicity (CSH)growth
devices. Thethe
and C. albicans
offilamentous
research significantly
groupgrowthobserved decreased
of C.that coumarin
albicans after
notcoumarin
significantly only treatment,
suppresses
decreased after
indicating
biofilm
coumarin that
formation coumarin
but also
treatment, inhibits
affects the
indicating biofilm
thatstructureformation
coumarin of inhibitsby suppressing
the mature biofilm;
biofilm adhesion
moreover
formation [100].
by the adhesion,adhesion
suppressing the cell
[100]. The
surface antifungal potency
hydrophobicity (CSH) and of coumarin-based
the filamentous growth triazoles against
of C. other
albicans fungal strains
significantly in addition
decreased after
to Candida
coumarin species indicating
treatment, had been that evaluated
coumarin by Dharavath and colleagues
inhibits biofilm formation by in suppressing
2020. Coumarin-based
adhesion
1,4-disubstituted 1,2,3-triazole derivatives were synthesized using a highly efficient, eco-friendly
[100].
protocol via a copper(I)-catalyzed click reaction between various substituted arylazides and terminal
alkynes. The in vitro antifungal activity was tested against Aspergillus niger, Aspergillus flavus and
The antifungal potency of coumarin-based triazoles against other fungal strains in addition to
Candida species had been evaluated by Dharavath and colleagues in 2020. Coumarin-based 1,4-
disubstituted 1,2,3-triazole derivatives were synthesized using a highly efficient, eco-friendly
Int. J. Mol. Sci. 2020, 21, 4618 17 of 81
protocol via a copper(I)-catalyzed click reaction between various substituted arylazides and terminal
alkynes. The in vitro antifungal activity was tested against Aspergillus niger, Aspergillus flavus and
Fusarium oxysporum by using the disc diffusion method and the results were compared with those of
clotrimazole, the reference drug. Compounds
clotrimazole, Compounds 63-68 (Figure 26),
63–68 (Figure 26), characterized
characterized by
by the
the presence
presence of a
para-substituted phenyl
para-substituted phenyl moiety, directly linked to the triazole ring, showed comparable activity in
respect to the reference compound clotrimazol [101,102].
[101,102].
Coumarin-based 1,4-disubstituted 1,2,3-triazole derivatives.

Figure 26. Coumarin-based
2.6. Antiviral
2.6. Antiviral Activity
Activity
2020 has
2020 has been
been aa crucial
crucial year
year for
for the
the timeless
timeless war war between
between human
human and and viruses: World Health
viruses: World Health
Organization (WHO) declared the outbreak of Sars-COVID-19
Organization (WHO) declared the outbreak of Sars-COVID-19 a Public Health Emergency of a Public Health Emergency of
International Concern
International Concern on on 30
30 January
January 20202020 and
and on on 1111 March
March WHO WHO characterized COVID-19 as
characterized COVID-19 as aa
pandemic [103]. Whole developed countries have been quarantined
pandemic [103]. Whole developed countries have been quarantined and generations that never faced and generations that never faced
medical crisis
medical crisis are
are now
now struggling
struggling withwith the
the consequences
consequences of of the
the viral
viral diffusion. Human history
diffusion. Human history is is
afflicted by
afflicted bythe thecyclic
cyclic succession
succession of pandemic
of pandemic events and the
events andresearch of newof
the research antivirals is still ongoing,
new antivirals is still
due to the ability of viruses to mutate and the continuous appearance
ongoing, due to the ability of viruses to mutate and the continuous appearance of new viruses of new viruses on the medical
on the
scenario [104].
medical scenario [104].
The natural
The natural worldworld is is a
a priceless
priceless source
source of of valuable
valuable medical compounds and
medical compounds and also
also in
in the
the fight
fight
against viral diseases there are several natural molecules which exhibit
against viral diseases there are several natural molecules which exhibit antiviral activity [105–109]. antiviral activity [105–109].
Coumarins, likewise
Coumarins, likewise otherother polyphenolic
polyphenolic compounds,
compounds, exert exert aa remarkable
remarkable antiviral
antiviral activity
activity [110,111].
[110,111].
The antiviral activity of coumarins explicates through different mechanisms
The antiviral activity of coumarins explicates through different mechanisms which affect the life which affect the life cycle
cycle
of viruses and their biological activities could be changed depending
of viruses and their biological activities could be changed depending upon the combination of upon the combination of various
substituents
various and conjugates
substituents [104,112].
and conjugates Coumarins
[104,112]. appears
Coumarins to be active
appears against
to be active several
against viruses,
several like
viruses,
HIV, influenza, hepatitis, Dengue and Chikunguya
like HIV, influenza, hepatitis, Dengue and Chikunguya [104]. [104].
Liu and
Liu and co-workers
co-workers after after aa phytochemical
phytochemical study study on on the
the stem
stem of Clausena lenis
of Clausena lenis isolated
isolated three
three new
new
and nine
and nine known
known prenylated
prenylatedcoumarins
coumarins[113]. [113].AllAll
thethe prenylated
prenylated coumarins
coumarins were were evaluated
evaluated bothboth
for
their anti-inflammatory and anti-HIV reverse transcriptase (RT) activities. In this last case, the
for their anti-inflammatory and anti-HIV reverse transcriptase (RT) activities. In this last case, the
inhibition assay
inhibition assay for the cytopathic
for the cytopathic activities
activities ofof HIV-1
HIV-1 (EC(EC50 ) as well as cytotoxic activity assay against
50) as well as cytotoxic activity assay against
C8166 cell line (CC 50 ) according to MTT methods
C8166 cell line (CC50) according to MTT methods were applied [114,115]. were applied [114,115].
The three new isolated compounds (69–71,
The three new isolated compounds (69–71, Figure 27) showed Figure 27) showed thethe
best
bestinhibitory
inhibitory activity
activity with
withan
EC50
an ECof 0.29, 0.68 and 0.17 µM, respectively. Furthermore, no cytotoxicity was observed against C8166
50 of 0.29, 0.68 and 0.17 μM, respectively. Furthermore, no cytotoxicity was observed against
cell
Int. line (CC
J. Mol.cell
C8166 Sci. 50 >
2020,
line 21,200.00
(CC x 50
FOR µM).REVIEW
PEER
> 200.00 μM). 18 of 83
Figure 27. New prenylated coumarins from Clausena lenis.

Figure 27. New prenylated coumarins from Clausena lenis.
Prenylated coumarins came to the fore in 2019 thanks to a work by Liu et al., who revealed for
the first time the presence of these type of derivatives in the fruits of Manilkara zapota, an ever-green
tropical tree. Also in this case, three new derivatives were identified (72–74, Figure 28) together with
seven known compounds and the team evaluated their anti-inflammatory and anti-HIV activities,
exploiting the methods described above [116].
Int. J. Mol. Sci. 2020, 21, 4618 18 of 81
Figure 27.
Figure 27. New
New prenylated
coumarins from
from Clausena
Clausena lenis.
lenis.
Prenylated coumarins came

Prenylated came to thethe fore in
in 2019 thanks
thanks to aa work
work by LiuLiu et al.,
al., who
who revealed
revealed for
Prenylated coumarins
coumarins came to to the fore
fore in 2019
2019 thanks toto a work by by Liu et
et al., who revealed for
for
the
the first time the presence of these type of derivatives in the fruits of Manilkara zapota, an ever-green
the first time the
first time presence of
the presence of these
these type
type of
of derivatives
derivatives in
in the
the fruits
fruits of Manilkara zapota,
of Manilkara zapota, an
an ever-green
ever-green
tropical tree.
tropical tree. Also
Also in this
this case, three
three new derivatives
derivatives were identified
identified (72–74, Figure
Figure 28) together
together with
tropical tree. Also in
in this case,
case, three new
new derivatives were
were identified (72–74,
(72–74, Figure 28)
28) together with
with
seven known compounds
seven compounds and the the team evaluated
evaluated their anti-inflammatory
anti-inflammatory and anti-HIV
anti-HIV activities,
seven known
known compounds and and the team
team evaluated their
their anti-inflammatory and and anti-HIV activities,
activities,
exploiting the
exploiting the methods
methods described
described above
above [116].
[116].
exploiting the methods described above [116].
HO
HO HO
HO
O
O O
O O
O
O
O O
O O
O O
O O
O O
O O
O O
O O
O
72
72 73
73 74
74
Figure 28.
Figure 28. Prenylated coumarins
28. Prenylated
Prenylated from
coumarins from Manilkara
from Manilkara zapota.
Manilkara zapota.
zapota.
Figure coumarins
The
The newnew
new prenylated coumarins
coumarins showed showed
showed the the highest
the highest anti-HIV
highest anti-HIV
anti-HIV RT RT effect
effect among
RT effect among the the prenylated
prenylated
The prenylated among the prenylated
coumarins
coumarins derived
derived from
from the
the fruit
fruit of
of Manilkara
Manilkara zapota;
zapota; in
in particular,
particular, compound
compound 72
72 displayed
displayed the most
the most
most
coumarins derived from the fruit of Manilkara zapota; in particular, compound 72 displayed the
powerful
powerful effect
effect with
with an
an EC
EC 50
50
value
value of
of 0.12
0.12 µM.
μM. Comparative
Comparative studies
studies between
between compounds
compounds 72–74
72-74 and
and
powerful effect with an EC50 value of 0.12 μM. Comparative studies between compounds 72-74 and
the other
the other coumarin
other coumarin derivatives
coumarin derivatives isolated
derivatives isolated
isolated fromfrom
from thethe fruits
the fruits highlighted
fruits highlighted
highlighted the the importance
the importance
importance of of the
of the isopentenyl
isopentenyl
theisopentenyl
the
group
group as
as aa substituent
substituent at
at C6
C6 and
and 2-methylbut-3-en-2-yl
2-methylbut-3-en-2-yl group
group as
as aa substituent
substituent at
at C3
C3 for
for the
the anti-HIV
anti-HIV
group as a substituent at C6 and 2-methylbut-3-en-2-yl group as a substituent at C3 for the anti-HIV
RT effect.
RT effect.
effect.
RT
In
In the
thesamesameyear,year,Jesumoroti
Jesumoroti andandco-workers
co-workers approached
approached the theproblem
problemfromfrom another pointpoint
another of view, of
In the same year, Jesumoroti and co-workers approached the problem from another point of
both
view, regarding
both the
regarding target
the and
target the
and origin
the of
origin the
of coumarin
the coumarin derivatives
derivatives [117].
[117]. First
First of
of all,
all, aa different
different
view, both regarding the target and the origin of the coumarin derivatives [117]. First of all, a different
viral
viral target
target was was chosen,
chosen, HIV-1
HIV-1 IN, IN, which
which is is essential
essential for for aaa stable
stable infection. Moreover,
infection. Moreover,
Moreover, there there are
are no
there are no
viral target was chosen, HIV-1 IN, which is essential for stable infection. no
homologous
homologous enzymes
enzymes in
in the
the host
host cell
cell [118].
[118]. Secondarily,
Secondarily, the
the coumarin
coumarin derivatives
derivatives were
were obtained
obtained
homologous enzymes in the host cell [118]. Secondarily, the coumarin derivatives were obtained
decorating
decorating the the coumarin
the coumarin nucleus
coumarin nucleus
nucleus with with
with an an hydrazide
an hydrazide
hydrazide group, group, in
group, order
in order
order toto achieve
achieve aaa synergistic
to achieve synergistic
synergistic effecteffect
effect
decorating in
against-HIV-1-IN
against-HIV-1-IN and and reducing
and reducing
reducing the the toxicity
the toxicity correlated
toxicity correlated
correlated to to the
to the salicylhydrazide,
the salicylhydrazide,
salicylhydrazide, which which
which was was however
was however
however
against-HIV-1-IN
essential
essential for
for the
the activity
activity [119–121].
[119–121]. The
The synthesized
synthesized compounds
compounds were
were evaluated
evaluated for
fortheir
their ininvitro
vitro HIV-1
HIV-
essential for the activity [119–121]. The synthesized compounds were evaluated for their in vitro HIV-
IN
1 IN inhibitory
inhibitory activity
activityusing
using chicoric
chicoric acid
acid (CA)
(CA) asasa areference
reference compound,
compound, according
according to
to the
the method
method
1 IN inhibitory activity using chicoric acid (CA) as a reference compound, according to the method
described
described by by McColl
McColl et
by McColl et al.
al. [122].
[122].
described et al. [122].
Compounds
Compounds 75–78
75–78 (Figure29)29)
(Figure appeared
appeared to beto the
be be most
the mostactive active
amongamong the whole
wholetheseries
whole series
showing
Compounds 75–78 (Figure 29) appeared to the most active among the series showing
showing
an inhibition
inhibitionan inhibition of extracellular
of extracellular
extracellular IN (evaluated
(evaluated IN (evaluated
in vitro in vitrotoaccording
vitro according
according the method
method to described
the method by described
McColl et et
an of IN in to the described by McColl
by
al. McColl
[122]) in et
a al. [122])
range fromin a
95% rangeto 86from
% and95% IC to
50
86 %
between and 13ICnM 50 between
(compound 13 nM
76) (compound
and 31 nM (CA and
76) IC 50
31
=10
al. [122]) in a range from 95% to 86 % and IC50 between 13 nM (compound 76) and 31 nM (CA IC50=10
nM
nM).(CA IC50 = 10 nM).
Furthermore, Furthermore,
the cytotoxicity
cytotoxicity of the cytotoxicity
all the
the obtainedof all the obtained
compounds compounds
was tested
tested was tested75–78
and derivatives
derivatives and
nM). Furthermore, the of all obtained compounds was and 75–78
derivatives
showed low low75–78 showed cytotoxicity.
or negligible
negligible low or negligible cytotoxicity.
showed or cytotoxicity.
Figure 29.
Figure Coumarin derivatives
29. Coumarin derivatives synthesized
synthesized by
by Jesumoroti
Jesumoroti and co-workers [117].
and co-workers
co-workers [117].
[117].
Seasonal influenza claims around 0.29–0.65 million victims annually, even if several drugs are
commercially available [123]. The flu is a contagious respiratory disease caused by influenza viruses,
manifesting major epidemics with no predictable periodicity or pattern and all different from one to
another [124]. For these reasons, a constant effort in the development of new drugs for the treatment of
this disease is of greatest importance. In 2019 Osman and co-workers combined two bioactive moieties
into a single molecule in order to obtain new bioactive compounds with antibacterial and antiviral
effects [125]. In particular, based on a previous study of the same research team, in which coumarin
scaffolds and thiazole moiety were combined leading to compounds endowed with both antibacterial
commercially available [123]. The flu is a contagious respiratory disease caused by influenza viruses,
manifesting major epidemics with no predictable periodicity or pattern and all different from one to
another [124]. For these reasons, a constant effort in the development of new drugs for the treatment
of this disease is of greatest importance. In 2019 Osman and co-workers combined two bioactive
Int. J. Mol. Sci. 2020, 21, 4618 19 of 81
moieties into a single molecule in order to obtain new bioactive compounds with antibacterial and
antiviral effects [125]. In particular, based on a previous study of the same research team, in which
coumarin
and scaffolds
antiviral andthe
activities, thiazole moiety
potentiality werecombination
of this combined leading to compounds
was further endowed
explored [126], [127]. with
Four both
new
antibacterial and antiviral activities, the potentiality of this combination was further
molecules of the series showed a remarkable antiviral activity against H1N1 virus. Compounds 79–82 explored [126],
[127]. Four
(Figure new molecules
30) seemed of the series
to be promising showed
agents, a remarkable
having IC50 valuesantiviral
of 4.84,activity against
19.72, 6.12 and H1N1 virus.
9.13 µg/mL,
Compounds 79–82 (Figure
respectively, against the H1N1 virus.30) seemed to be promising agents, having IC 50 values of 4.84, 19.72, 6.12
and 9.13 μg/mL, respectively, against the H1N1 virus.
Figure 30. Hybrid

Figure 30. Hybrid pharmacophores
pharmacophores proposed
proposed by
by Osman
Osman and
and co-workers
co-workers [125].
[125].
A similar approach was followed by Pavurala et al., in 2019, who exploited the versatility of the
A similar approach was followed by Pavurala et al., in 2019, who exploited the versatility of the
coumarin scaffold by combining it with another bioactive moiety, in this case a triazolothiadiazine [128].
coumarin scaffold by combining it with another bioactive moiety, in this case a triazolothiadiazine
The result was the synthesis of a series of bis-coumarinyl-bis-triazolothiadiazinyl ethane derivatives
[128]. The result was the synthesis of a series of bis-coumarinyl-bis-triazolothiadiazinyl ethane
that were evaluated for their antiviral activities against different DNA and RNA viruses. Among the
derivatives that were evaluated for their antiviral activities against different DNA and RNA viruses.
tested compound 83 (Figure 31) exhibited a good antiviral activity against influenza A virus. More
Among the tested compound 83 (Figure 31) exhibited a good antiviral activity against influenza A
specifically, in cytopathic effect (CPE) reduction assay against influenza virus infected Madin–Darby
virus. More specifically, in cytopathic effect (CPE) reduction assay against influenza virus infected
canine kidney (MDCK) cells, it displayed antiviral EC50 values of 20–72 µM.
Madin–Darby canine kidney (MDCK) cells, it displayed antiviral EC50 values of 20–72 μM.
S N
N
N
N OH O O
O O HO N
N
N
N S
83
Figure 31.
Figure 31. Bis coumarinyl bis triazolothiadiazinyl ethane
ethane derivative.
derivative.
Bizzarri
Bizzarri etetal. al.
exploited the regioselective
exploited oxidation
the regioselective of coumarin
oxidation of derivatives
coumarin with 2-iodoxybenzoic
derivatives with 2-
acid (IBX) in order
iodoxybenzoic acidto obtain
(IBX) catechol
in order and pyrogallol
to obtain catechol andmoieties [129].moieties
pyrogallol After cytotoxicity
[129]. After studies that
cytotoxicity
confirmed
studies thatthe safety ofthe
confirmed thesafety
seriesofof
thederivatives, the antiviral
series of derivatives, the activity
antiviralagainst
activityinfluenza viruses
against influenza
A/PR8/H1N1 was evaluated
viruses A/PR8/H1N1 and compounds
was evaluated 84–89 (Figure
and compounds 32) resulted
84–89 (Figure able toable
32) resulted inhibit the viral
to inhibit the
replication. Interestingly, pyrogallol derivatives
viral replication. Interestingly, pyrogallol derivatives 885088 (IC = 69.9 µg/mL) and 89
(IC50 = 69.9 μg/mL) and 89 (IC(IC =
5050 = 47.9 µg/mL)
μg/mL)
turned
turnedout
outtotobebe
moremore active thanthan
active catechol derivatives
catechol derivatives 50 =
84 (IC84 106.5
(IC 50 = µg/mL) and 85 (IC
106.5 μg/mL) = 91.5
and5085 (IC50µg/mL).
= 91.5
Moreover, pyrogallol and
μg/mL). Moreover, catechol derivatives
pyrogallol and catecholwere more active than
derivatives werethe more
monohydroxycoumarins
active than the
6-hydroxycoumarin
monohydroxycoumarins and 7-hydroxycoumarin
6-hydroxycoumarin(IC 50 110
and µg/mL for both the
7-hydroxycoumarin (ICparent compounds).
50 110 μg/mL for both the
parent compounds).
Int. J. Mol. Sci. 2020, 21, 4618 20 of 81
Figure 32. Antiviral coumarins from Bizzarri et al. [129].

Figure 32. Antiviral coumarins from Bizzarri et al. [129].
One of the possibleFigure 32. Antiviral
advantages coumarins
of oxidized from Bizzarri
coumarins couldet al.
be[129].
their mode of action against
OneIndeed,
viruses. of the possible
due toadvantages of oxidized
their antioxidant coumarins
activity, could be
coumarins their mode
derivatives of action
could against
affect viruses.
intracellular
One
Indeed, of the possible
due to their advantages
antioxidant of oxidized coumarins could be their mode of action against
redox-sensitive pathways usefulactivity, coumarins
for viral derivatives
replication, could affect
independently from intracellular
the variabilityredox-sensitive
of the strains
viruses.
pathways Indeed,
useful due
for to their
viral antioxidant
replication, activity, coumarins
independently from the derivatives
variability of could
the affect[129].
strains intracellular
[129].
redox-sensitive
As alreadypathways
mentioned, useful for viral replication,
coumarins have been independently
studied from thepotential
variabilityapplication
of the strains
As already mentioned, coumarins have been studied alsoalsofor for
theirtheir
potential application as
as anti-
[129].
anti-hepatitis agents.
hepatitis agents. Tsay Tsay and co-workers
and co-workers studied
studied the activity
the activity against against hepatitis
hepatitis C virusC virus
(HCV)(HCV) of someof
some As already mentioned,
unnatural coumarins
imidazole-coumarin have been
conjugates studied
[130]. Abovealsoall,
forthree
theircompounds
potential application
(90–92, as anti-
Figure 33)
unnatural imidazole-coumarin conjugates [130]. Above all, three compounds (90–92, Figure 33)
hepatitis
showed agents. Tsay antiviral
and co-workers studied the activityEC against hepatitis
rangingCfrom
virus5.1
(HCV)
to 8.4 of some
showed aa noteworthy
noteworthy antiviral activity
activity against
against HCV
HCV with
with EC50 values
50 values ranging from 5.1 to 8.4 μM and
µM and
unnatural imidazole-coumarin conjugates [130]. Above all, three compounds (90–92, compound33)
Figure
selective
selective indices (SI =
indices (SI CC50
= CC /EC50, ,which
50/EC50
whichisisaameasure
measure for for the
the therapeutic
therapeutic window
window of of the
the compound in
in
showed
an assay asystem)
noteworthy antiviral
higher than activity against HCV with EC50 values ranging from 5.1 to 8.4 μM and
20.
an assay system) higher than 20.
selective indices (SI = CC50/EC50, which is a measure for the therapeutic window of the compound in
an assay system) higher than 20.
Figure
Figure 33. Anti-hepatitis coumarins.
33. Anti-hepatitis coumarins.
Huang
Huang and
andco-workers
co-workersfocused their
Figure
focused attention
33.
their ononthe
Anti-hepatitis
attention investigation
coumarins.
the of of
investigation thethe
potentiality expressed
potentiality by
expressed
esculetin (or (or
by esculetin 6,7-dihydroxycoumarin)
6,7-dihydroxycoumarin) against hepatitis
against B virus
hepatitis (HBV)
B virus [131].[131].
(HBV) The The
results suggested
results that
suggested
Huang
esculetin and co-workers
efficiently inhibits focused
HBV their attention
replication both in on theand
vitro investigation
in vivo, of theprovides
which potentiality
an expressed
opportunity
that esculetin efficiently inhibits HBV replication both in vitro and in vivo, which provides an
by
for esculetin (or 6,7-dihydroxycoumarin)
further development of the compound against hepatitis B virus (HBV) [131]. The results suggested
opportunity for further development of theascompound
antiviral agent.
as antiviral agent.
that esculetin efficiently inhibits HBV replication both in vitro and in vivo, which provides an
2.7. Anti-Inflammatory
opportunity Activity
for furtherActivity
development of the compound as antiviral agent.
2.7. Anti-Inflammatory
Inflammation is a general physiological response that aims, firstly, to the circumscription of the
2.7. Inflammation is aActivity
Anti-Inflammatory general physiological response that aims, firstly, to the circumscription of the
harmful factors, which could be both endogenous (e.g., cancer, ischemia, autoimmune diseases) and
harmful factors, which could be both endogenous (e.g., cancer, ischemia, autoimmune diseases) and
exogenous (e.g., viral
Inflammation is aor bacterial
general infections, trauma),
physiological response secondarily, to theto
that aims, firstly, removal of the causes of
the circumscription of the
the
exogenous (e.g., viral or bacterial infections, trauma), secondarily, to the removal of the causes of the
damage factors,
harmful and finally
whichto could
the reparation of the tissues
be both endogenous andcancer,
(e.g., restoration of the
ischemia, functions. diseases)
autoimmune Nevertheless,
and
damage and finally to the reparation of the tissues and restoration of the functions. Nevertheless, the
the inflammation
exogenous andorthe
(e.g., viral consequent
bacterial restorative
infections, trauma), process could become
secondarily, harmful
to the removal of for
the the organism
causes of the
inflammation and the consequent restorative process could become harmful for the organism itself
itself when
damage andthere is to
finally a persistent stimulation
the reparation and the
of the tissues andphases of inflammation
restoration and reconstruction
of the functions. Nevertheless, the are
when there is a persistent stimulation and the phases of inflammation and reconstruction are
contemporaryand
inflammation activated, causing tissue
the consequent injuries
restorative and fibrosis
process [132]. During
could become harmful an for
inflammatory
the organism process,
itself
contemporary activated, causing tissue injuries and fibrosis [132]. During an inflammatory process,
many inflammatory
when effectorsstimulation
there is a persistent and mediators andare theproduced
phases of andinflammation
involved, often andwith a common effect
reconstruction are
many inflammatory effectors and mediators are produced and involved, often with a common effect
on vascular system
contemporary and on
activated, the recruitment
causing of leukocytes
tissue injuries and fibrosis[133]. Frequently,
[132]. During aninflammatory
inflammatorymediators
process,
on vascular system and on the recruitment of leukocytes [133]. Frequently, inflammatory mediators
are theinflammatory
many target of anti-inflammatory drugs [134–137].
effectors and mediators are produced and involved, often with a common effect
are the target of anti-inflammatory drugs [134–137].
Amongsystem
on vascular the numerous
and on biological activities
the recruitment shown by coumarin
of leukocytes derivatives,
[133]. Frequently, the anti-inflammatory
inflammatory mediators
effect
are thecould
targetnot
of certainly miss. Dawood
anti-inflammatory drugset[134–137].
al., in 2015, developed a new series of coumarin derivatives
hybridizing two pharmacophoric moieties—the coumarin scaffold itself and thiazoline or thiazolidinone
groups, both showing cyclooxygenase 2 (COX-2) inhibitor effect [127], [138–143]. The compounds were
Among the numerous biological activities shown by coumarin derivatives, the anti-
inflammatory effect could not certainly miss. Dawood et al., in 2015, developed a new series of
coumarin
Int. J. Mol. Sci. derivatives
2020, 21, 4618 hybridizing two pharmacophoric moieties—the coumarin scaffold itself21and of 81
thiazoline or thiazolidinone groups, both showing cyclooxygenase 2 (COX-2) inhibitor effect [127],
[138–143]. The compounds were evaluated in vivo for their systemic effect, in vitro for their ability to
evaluated in vivo for their systemic effect, in vitro for their ability to inhibit human COX-1 and COX-2
inhibit human COX-1 and COX-2 and also to evaluate the ulcerogenic potential compared to the
and also to drug,
reference evaluate the ulcerogenic
indomethacin, always potential
followingcompared
standardtomethods
the reference drug,
reported indomethacin,
in literature. Most always
of the
following
new compoundsstandard showed
methodssignificant
reported ininliterature. Most of the new
vivo anti-inflammatory compounds
activity with ashowed significant
superior gastro-
inintestinal
vivo anti-inflammatory activityulceration)
safety profiles (0–7% with a superior gastro-intestinal
as compared safety profiles
to indomethacin. The IC(0–7% ulceration) as
50 values of all the
compared to indomethacin.
bioactive compounds ranged between The IC50 values of all the bioactive compounds ranged between
0.31 and 0.78 mM, showing an in vitro high affinity 0.31and
and
0.78 mM, showing
selectivity towardan inthe
vitro high affinity
COX-2 and selectivity
isoenzyme, comparedtoward
to the
theCOX-2 isoenzyme,
reference compared
celecoxib. Ethylto
the reference celecoxib. Ethyl thiosemicarbazone 93, thiazoline derivatives 94–98
thiosemicarbazone 93, thiazoline derivatives 94–98 and the thiazolidinone compounds 99–100 and the thiazolidinone
compounds
exhibited the 99–100 exhibited
highest in vivothe
andhighest
in vitroinanti-inflammatory
vivo and in vitro anti-inflammatory
activities with goodactivities with good
COX-2 selectivity
COX-2
(Figure selectivity
34) [143].(Figure 34) [143].
Figure 34. Anti-inflammatory

Figure34. coumarin derivatives.
Anti-inflammatory coumarin derivatives.
Nevertheless,
Nevertheless,cyclooxygenases
cyclooxygenases are are not
not the only enzymes
the only enzymesinvolved
involvedininthe theinflammatory
inflammatoryprocess.
process.
Actually, 5-lipoxygenase (5-LOX) catalyzes two different steps in the arachidonic
Actually, 5-lipoxygenase (5-LOX) catalyzes two different steps in the arachidonic acid metabolism acid metabolism
that
thatbrings
bringstotothe
theproduction
production of leukotrieneAA4,4which
of leukotriene , whichis is successively
successively metabolized
metabolized intointo leukotriene
leukotriene B4
B4[144].
[144]. Molecular
Molecular inhibitors
inhibitors of leukotrienes
of leukotrienes competitively
competitively bind thebind thesite
active active site ofand
of 5-LOX 5-LOX and are
are divided
divided
in threeincategory:
three category: redox-active
redox-active compounds compounds (i.e., coumarins),
(i.e., coumarins), iron-ligand
iron-ligand inhibitorsinhibitors
with weak with weak
redox-
redox-active properties
active properties and non-redox-type
and non-redox-type inhibitors
inhibitors [145]. [145].
InIn2016,
2016,Srivastava
Srivastavaandandcolleagues
colleagues evaluated
evaluated thethe
anti-inflammatory
anti-inflammatory andand
analgesic effect
analgesic of a series
effect of a
ofseries
synthesized 7-substituted
of synthesized coumarins
7-substituted and, consequently,
coumarins the mostthe
and, consequently, active
mostcompounds were assessed
active compounds were
inassessed
vitro forin 5-LOX
vitro inhibition
for 5-LOX [146]. Compounds
inhibition 101 and 102
[146]. Compounds 101(Figure
and 102 35)(Figure
resulted
35)the most promising
resulted the most
promising derivatives,
derivatives, also in the ulcerogenic
also in the ulcerogenic risk evaluation
risk evaluation when comparedwhen compared to acetylsalicylic
to acetylsalicylic acid. Inacid.
vitro
In vitro
kinetic kinetic
study of compound 102 (IC50 =
study of compound 102 (ICnM)
2.09 50 = 2.09 nM)mixed
showed showed or mixed or non-competitive
non-competitive type of
type of inhibition
ofinhibition
5-LOX. The of presence
5-LOX. The of apresence
substituentof aon substituent
C6 position onofC6 position of benzothiazole
benzothiazole ring was found ring
verywas found
important
very
for important
increasing thefor increasing the activity.
activity.
Int. J.
Int. J. Mol.
Mol. Sci.
Sci. 2020,
2020, 21,
21, x4618
FOR PEER REVIEW 22 of
22 of 81
83
Figure 35.
Figure 35. Anti-inflammatory
Anti-inflammatory 7-substituted
7-substituted coumarins
coumarins proposed
proposed by
by Srivastava
Srivastava [146].
[146].
Figure 35. Anti-inflammatory 7-substituted coumarins proposed by Srivastava [146].
In 2018,
In 2018, Liu
Liuetetal.al.identified
identifiedten tennew
newcoumarin
coumarinderivatives
derivatives (3(3 monomers
monomers andand 7 dimers)
7 dimers) isolated
isolated in
in aIn 2018, Liu
phytochemical et al. identified
study on ten
Murraya new coumarin
exotica, a derivatives
dwarf tree that (3
is monomers
native of theand 7 dimers)
tropical
a phytochemical study on Murraya exotica, a dwarf tree that is native of the tropical and subtropical and isolated
subtropical
inareas
a phytochemical
areas of Asia
of Asia and study on Murraya
and traditionally
traditionally used exotica,
used a dwarf tree
treatment
in the treatment that is native of the tropical
inflammatory-related
of inflammatory-related diseases
diseasesand subtropical
[147].
[147]. Previous
Previous
areas of
studies Asia
had and
shown
studies had shown that traditionally
that the used
main in the
active treatment
components
components of inflammatory-related
in M. exotica were diseases
coumarins; [147].
also Previous
bis-coumarins
bis-coumarins
studies
were had shown
were isolated
isolated from
from thatthethe
the mainAactive
plant.
plant. components
further
further in M.
investigation
investigation onexotica
the 95% were coumarins;
aqueous
aqueous EtOHalso bis-coumarins
extract of the
the roots
roots
were isolated
allowed
allowed the from the plant.
the obtainment
obtainment A further
of new
of new investigation
coumarins,
coumarins, on
together with
together the
with 95%bioactive
other
other aqueous molecules.
bioactive EtOH extract
molecules. Theofcompounds
The the roots
compounds
allowed
were the
tested obtainment
for their of new
inhibitorycoumarins,
effect on together
NO with
productionother
andbioactive
compound
were tested for their inhibitory effect on NO production and compound 103 (Figure 36) stood molecules.
103 The
(Figure compounds
36) stood out
out
were tested
among the
among for their
the coumarins
coumarins in inhibitory
in the effect
the inhibition on NO
inhibition against production
against LPS-induced and
LPS-induced NO compound
NO production 103
production in(Figure
in BV-2 36) stood
BV-2 microglial out
microglial cells
cells
among
with IC
with the
IC coumarins
50 value
50 value of
of 8.6
8.6 in the
μM.
µM. inhibition against LPS-induced NO production in BV-2 microglial cells
with IC50 value of 8.6 μM.
Figure 36. One of the coumarin derivatives isolated from M. exotica.

Figure 36. One of the coumarin derivatives isolated from M. exotica.
Figure 36. (NF-kB)
The nuclear factor-kB One of thefamily
coumarin derivatives isolated
of transcription from
factors is M. exotica. by a set of related,
composed
The nuclear
evolutionarily factor-kB
conserved (NF-kB) family
DNA-binding of transcription
proteins. In response to factors is composed
multiple stimuli such by asa set of related,
inflammatory
The nuclear
evolutionarily factor-kB
conserved (NF-kB) family
DNA-binding of transcription
proteins. In factors
responseis composed
to multiple
cytokines, bacterial lipopolysaccharide (LPS), viral infection or stress, a series of cascade reactions by a set
stimuliof related,
such as
evolutionarily
inflammatory conserved
cytokines, DNA-binding
bacterial proteins.
lipopolysaccharide In response
(LPS), viral
bring towards the entry on NF-kB into the nucleus and to the activation of several genes to multiple
infection or stimuli
stress, asuch
series asof
inflammatory
cascade reactions
participating cytokines, bacterial
bring towards
in immune lipopolysaccharide
the entry on
and inflammatory NF-kB into
responses, (LPS),
cell the viral infection
nucleusgrowth
adhesion, or
and tocontrol stress,
the activation a series ofof
of several
and regulation
cascade
genes reactions
participating
apoptosis bring
[148,149]. intowards
In immune
2019, Fanthe
andentry
and on NF-kB
inflammatory
co-workers into the innucleus
responses,
evaluated vivocelland
and intovitro
the activation
adhesion, thegrowth of several
control
anti-inflammatory and
genes participating
regulation
activity of osthole in
of apoptosis immune
(Figure[148,149]. and
37) [150],In inflammatory
2019, Fan
a natural responses,
and co-workers
prenylated coumarin cell adhesion,
evaluated
from Cnidiumin vivo growth control
and in vitro
Monnieri and
the anti-
(L.) Cuss. but
regulation of
inflammatory apoptosis
activity [148,149].
of In
osthole 2019,
(Figure Fan and
37) co-workers
[150], a naturalevaluated in
prenylated vivo
also found in other medicinal plants, which has already shown different biological and pharmacological and
coumarin in vitro
from the anti-
Cnidium
inflammatory
Monnieri (L.)
properties activity
suchCuss. of osthole
but also found
as neuroprotective, (Figure
in 37) [150],
other
osteogenic, a natural
medicinal plants,
immunomodulatory, prenylated hascoumarin
which anticancer, from Cnidium
alreadyhepatoprotective,
shown different
Monnieri (L.) Cuss. but
biological and pharmacological
cardiovascular also found in other
properties such
protective and antimicrobial medicinal plants,
as neuroprotective,
activities which has already
osteogenic, that
[13]. The team established shown different
immunomodulatory,
osthole inhibited
biological and
anticancer, pharmacological
hepatoprotective, properties
cardiovascular such as neuroprotective,
protective and osteogenic,
antimicrobial
the production of NO, PGE2, TNF-a and IL-6 in LPS-induced macrophages and suppressed immunomodulatory,
activities [13].theThe team
activity
anticancer,
established hepatoprotective,
that osthole cardiovascular
inhibited the protective
production of and
NO, antimicrobial
PGE2, TNF-a
of iNOS and COX-2, probably by blocking the activation of NF-kB and p38/MAPK signaling pathways. activities
and IL-6 [13].
in The team
LPS-induced
established
macrophages thatandosthole inhibited
suppressed thethe production
activity of iNOSofand NO,COX-2,
PGE2,probably
TNF-a and IL-6 in LPS-induced
by blocking the activation
macrophages
of NF-kB and and suppressed
p38/MAPK the activity
signaling of iNOS and COX-2, probably by blocking the activation
pathways.
of NF-kB and p38/MAPK signaling pathways.
Int. J. Mol. Sci. 2020, 21, 4618 23 of 81
Int.J.J.Mol.
Int. Mol.Sci.
Sci.2020,
2020,21,
21,xxFOR
FORPEER
PEERREVIEW
REVIEW 2323ofof8383
Figure37.
Figure 37.Structure
Structureofofthe
thenatural
naturalcoumarin
coumarinosthole.
osthole.
Figure 37. Structure of the natural coumarin osthole.
Inthe
In
In thesame
the sameyear,
same year,Mu
year, Muand
Mu andcolleagues
and colleaguessynthesized
colleagues synthesizedaaaseries
synthesized seriesof
series ofofeleven
eleven7-substituted
eleven 7-substitutedcoumarins
7-substituted coumarinsand
coumarins and
and
evaluatedtheir
evaluated
evaluated theiranti-inflammatory
their anti-inflammatoryactivities
anti-inflammatory activitiesand
activities andtheir
and theirability
their abilityto
ability totoexploit
exploitthe
exploit theNF-kB
the NF-kBpathway
NF-kB pathway[151].
pathway [151].All
[151]. All
All
the
the screened
screened compounds
compounds showed
showed a a relevant
relevant anti-inflammatory
anti-inflammatory activity.
activity.
the screened compounds showed a relevant anti-inflammatory activity. In the series, compound 104 In
In the
the series,
series, compound
compound 104
104
(Figure38)
(Figure
(Figure 38)
38)was was
was identified
identified
identified as aasas aand
hit a hit
hit and further
and
further further studies
studies studies
of ofof molecular
molecular molecular
docking were docking
docking wereconfirming
were
conducted conducted
conducted
confirming that 104 could bind to the active site (NLS Polypeptide) of NF-κB p65.
that 104 could bind to the active site (NLS Polypeptide) of NF-κB p65. Its binding affinity was affinity
confirming that 104 could bind to the active site (NLS Polypeptide) of NF-κB p65. Its
Its binding
binding affinity
further
was further confirmed by surface plasmon resonance (SPR) analysis. Moreover,
confirmed by surface plasmon resonance (SPR) analysis. Moreover, Western blot assay showedassay
was further confirmed by surface plasmon resonance (SPR) analysis. Moreover, Western
Western blot
blot assay
that
showed
showed
104 that104
that
remarkably 104 remarkably
remarkably
blocked blocked
blocked
the NF-κB theNF-κB
the
signaling NF-κB signaling
signaling
pathway pathwayininvitro.
pathway
in vitro. vitro.
Figure 38. 7-substituted coumarin as blockers of NF-κB signaling pathway.

Figure38.
Figure 38.7-substituted
7-substitutedcoumarin
coumarinasasblockers
blockersofofNF-κB
NF-κBsignaling
signalingpathway.
pathway.
2.8. Neuroprotection: Effect on Alzheimer’s Disease
2.8.Neuroprotection:
2.8. Neuroprotection:EffectEffecton onAlzheimer’s
Alzheimer’sDiseaseDisease
Alzheimer’s disease (AD) is the most common form of dementia (60–70% of cases of dementia are
Alzheimer’s
causeAlzheimer’s
by AD) anddiseasedisease(AD)
consists (AD) isisthe
themost
mostcommon
in a neurodegenerative common formofof
form
disorder dementia(60–70%
dementia
characterized (60–70% ofofcases
by a slow, cases ofofdementia
dementia
progressive and
arecause
are causeby
irreversible by AD)
AD)
loss andconsists
of and
cognitiveconsists ininaaneurodegenerative
function neurodegenerative
and memory [152–157]. disorder
disorder characterized
The characterized
current therapeutic byaaslow,
by slow,progressive
approach,progressive
mainly
andirreversible
and
based irreversible
on the uselossloss ofofcognitive
cognitivefunction
of acetylcholinesterase function(AChE)andmemory
and memory [152–157].
[152–157].
inhibitors Thecurrent
The
(rivastigmine,current therapeutic
therapeutic
donepezil, approach,
approach,
galantamine)
ormainly
mainly based on
based
N-methy-D-aspartic on thetheacid
use(NMDA)
use ofof acetylcholinesterase
acetylcholinesterase
receptor inhibitors (AChE)
(AChE) inhibitors
inhibitors
(memantine), (rivastigmine,
is (rivastigmine,
merely symptomatic donepezil,
donepezil,
and
galantamine)
galantamine) or
or N-methy-D-aspartic
N-methy-D-aspartic acid
acid (NMDA)
(NMDA) receptor
receptor
does not counteract degeneration progression. New innovative approaches, such as multi-targeted inhibitors
inhibitors (memantine),
(memantine), is
is merely
merely
symptomatic
symptomatic
strategies, anddoes
and
are urgentlydoes notcounteract
not
required counteract degeneration
in orderdegeneration
to cure cognition progression.
progression.
and motor New
New innovative
innovative
dysfunctions, approaches,such
approaches,
neurodegeneration such
asasmulti-targeted
and multi-targeted
depression. Among strategies,
strategies, their arebiological
are urgentlyrequired
urgently required
activities, ininorder
ordertotocure
coumarins cure cognition
cognition
showed and
theand motor
motor
ability dysfunctions,
to dysfunctions,
inhibit some
neurodegeneration
neurodegeneration and
and depression.
depression. Among
Among their
their biological
biological activities,
activities,
biological targets involved in AD. With this in mind, some recent works aimed at investigating coumarins
coumarins showed
showed the
the ability
ability
to inhibit
to inhibit some
coumarins some biological
biological
potential targets involved
targets involved
in the treatment of AD are in
in AD. AD. With this in mind, some recent works aimed atat
With below.
discussed this in mind, some recent works aimed
investigating
investigating coumarins
coumarins
In 2019, Karakaya potential
potential
and ininthe
collaborators thetreatment
treatment
investigated ofofAD
AD
theareare discussedand
discussed
antioxidant below.
below.
AChE/BuChE inhibitory
InIn 2019,
2019, Karakaya
Karakaya and
and collaborators
collaborators investigated
investigated the
activity of aerial parts, fruits, flowers and root extracts from Ferulago cassiaAChE/BuChE
the antioxidant
antioxidant and
and AChE/BuChE
Boiss inhibitory
inhibitory
[158]. Root’s and
activity
activity
fruit’s ofofaerial
aerialparts,
dichloromethane parts,extracts
fruits,flowers
fruits, flowersand
showed andhighest
the rootextracts
root extracts fromFerulago
from
antioxidant Ferulagocassia
potential cassia
in TBA Boiss
Boiss
assay. [158].
[158]. Root’sand
The Root’s
evaluationand
fruit’s
fruit’s dichloromethane
dichloromethane extracts
extracts showed
showed the
the highest
highest antioxidant
antioxidant
of anticholinesterase activity was carried out by means of the Ellman’s method [159]: dichloromethane potential
potential inin TBA
TBA assay.
assay. The
The
evaluation
evaluation of
of anticholinesterase
anticholinesterase activity
activity was
was carried
carried out
out by
by means
means
extracts showed significant inhibition against BuChE (96.56% ± 2.98 and 82.33% ± 2.69, respectively) at of
of the
the Ellman’s
Ellman’s method
method [159]:
[159]:
dichloromethane
dichloromethane
20 extractsinhibition
extracts
µg/mL and appreciable showedsignificant
showed significant
against AChE inhibition
inhibition ±against
(53.24 against BuChE
BuChE
1.22 and (96.56%
31.38(96.56%
± 5.41%, ±±respectively)
2.98and
2.98 and82.33%
82.33%
at 20±±
µg/mL. Four coumarins were isolated from Ferulago cassia—peucedanol, suberosin, grandivitinol and±±
2.69,
2.69, respectively)
respectively) at
at 2020 μg/mL
μg/mL and
and appreciable
appreciable inhibition
inhibition against
against AChE
AChE (53.24
(53.24 ± ± 1.22
1.22 and
and 31.38
31.38
5.41%,respectively)
5.41%, respectively)
umbelliferone at20
(Figureat39). 20Therefore,
μg/mL.Four
μg/mL. Four coumarins
coumarins
F. cassia wereisolated
were
can be considered isolated fromFerulago
from
a starting Ferulago
point cassia—peucedanol,
forcassia—peucedanol,
the development of
suberosin,
suberosin, grandivitinol
grandivitinol and
and umbelliferone
umbelliferone
new compounds with antioxidant and anticholinesterase activity. (Figure
(Figure 39).
39). Therefore,
Therefore, F.
F. cassia
cassia can
can bebe considered
considered aa
starting point
starting point forfor the
the development
development ofof new new compounds
compounds with with antioxidant
antioxidant and and anticholinesterase
anticholinesterase
activity.
activity.
Int.
Int.J. J.Mol.
Mol.Sci.
Sci.2020,
2020,21,
21,x 4618
FOR PEER REVIEW 2424ofof8381
Figure 39. Coumarins extracted from Ferulago cassia.

Thanks
Thankstototheir theirsimple
simplestructural
structuralarchitecture
architectureand andchemical
chemicalstability,
stability,coumarins
coumarinscan canbe beeasily
easily
synthesized
synthesized and modified in order to produce more active and selective compounds. Najafi co-
Thanks and
to modified
their simple in order
structural to produce
architecturemore active
and and
chemical selective
stability, compounds.
coumarins Najafi
can be and
easily
and
workers
synthesized
co-workers synthesized
and
synthesized a series
modified ainseriesof tacrine-coumarin
order ofto produce morederivatives
tacrine-coumarin active and linked
derivatives selective
linked toto a a1,2,3-triazole
compounds. Najafi
1,2,3-triazole moiety
and and
moiety co-
and
evaluated
workers
evaluated their
theiractivity
synthesized ininterms
a series
activity of
of AChE
AChE and
of tacrine-coumarin
terms and butyrylcholinesterase
derivatives linked to(BuChE)
butyrylcholinesterase inhibition,
a 1,2,3-triazole
(BuChE) moiety
inhibition, keeping
and
keeping
donepezil
evaluated and
donepeziltheir andtacrine
tacrineas
activity reference
in
as terms ofdrugs
reference AChE
drugs [160].
andInIn
[160]. addition,
addition,their
theirbeta-secretase
butyrylcholinesterase (BuChE)11inhibition,
beta-secretase (BACE1)
(BACE1)inhibitory
keeping
inhibitory
activity
donepezil and
activity and neuroprotective
andneuroprotective
tacrine as reference potential were
drugswere
potential evaluated.
[160].evaluated.
In addition, Since tacrine
their
Since is a
beta-secretase
tacrine well-known
1 (BACE1)
is a well-known inhibitor ofofthe
inhibitory
inhibitor the
catalytic
activity site
and of AChE,
neuroprotectivewhereas coumarins
potential were showed
evaluated. affinity
Sincefor the
tacrine peripheral
is a
catalytic site of AChE, whereas coumarins showed affinity for the peripheral anionic site (PAS) [161], anionic
well-known site (PAS)
inhibitor [161],
of the
this
thisnew
catalyticnew compounds
site of AChE, may
compounds maybe
whereas bepotential
coumarins
potential dual—and therefore
showed affinity
dual—and therefore formore powerful
powerful-anionic
the peripheral
more -inhibitors
site of
inhibitors ofChEs.
(PAS)
ChEs. The
[161],
The
ininvitro
this newAChE
vitro AChEand
compounds andBuChEmay be
BuChE inhibitory
potentialactivity
inhibitory dual—and
activity was
wasevaluated
therefore
evaluatedmoreusing the
theEllman’s
usingpowerful method
- inhibitors
Ellman’s methodof [159];
ChEs.
[159]; among
The
among
all
in the
thetested
vitro
all testedmolecules,
AChE and BuChEcompound
molecules, inhibitory 105
compound 105resulted
activity was the
resulted the best
best in
evaluated AChE
in using
AChEthe inhibition
Ellman’s
inhibition (IC
(IC 5 0= 0.027
50 = 0.027
method ±±0.009
[159]; among
0.009 μM;
µM;
tacrine
alltacrine IC
the tested50 = 0.048 ± 0.011
= 0.048 ± 0.011
IC50 molecules, μM,
compound donepezil IC
105 resulted
µM, donepezil 50 = 0.039
= 0.039
IC50 the ± 0.097
best ±in0.097
AChEμM) and
inhibition
µM) compound
and compound 106
(IC5 0= 0.027 was
106 ±was the
0.009 most
theμM;most
promising
tacrine IC50 BuChE
promising 0.048 ±inhibitor
=BuChE 0.011 μM,
inhibitor (IC 50 = 0.006 ± 0.002
(ICdonepezil
50 = 0.006 ±IC
μM;
µM; tacrine
50 = 0.039
0.002 ± 0.097 IC
tacrine 50 = 0.010
50 =and
μM)
IC 0.010 ±±0.004
0.004μM,
compound µM,106donepezil
was the most
donepezil IC
IC5050 ==
8.416
promising± 0.628 μM)
BuChE (Figure
inhibitor
8.416 ± 0.628 µM) (Figure 40). 40).
(IC 50 = 0.006 ± 0.002 μM; tacrine IC 50 = 0.010 ± 0.004 μM, donepezil IC 50 =
8.416 ± 0.628 μM) (Figure 40).
Figure 40. (a) Chemical structure of compounds 105 and 106; panel (b) SAR studies.
Int. J. Mol. Sci. 2020, 21, 4618 25 of 81
Concerning
Concerning thethe anti-BuChE
anti-BuChE activity,
activity, structure-activity
structure-activity relationship
relationship studies
studies showed
showed that
that Cl Cl
andand
Me substituents, as well as the methylene linker, play a complex and
Me substituents, as well as the methylene linker, play a complex and not completely understood role not completely understood
in role in the enzyme
the enzyme inhibition. inhibition. From the of
From the evaluation evaluation
inhibitoryofactivity
inhibitory
of theactivity of thecompounds
synthesized synthesized
oncompounds on BACE1,
BACE1, a moderate a moderate
inhibitory activityinhibitory
of compoundactivity ofwas
105 compound
observed 105 was observed
(inhibition (inhibition
of 28.69% and
of 28.69% and 13.97% at 50 and 10 µM, respectively). Nevertheless,
13.97% at 50 and 10 μM, respectively). Nevertheless, compound 105 did not show neuroprotective compound 105 did not show
neuroprotective
action on PC12 cellsaction
exposed on to
PC12 cells
Aβ25-35 exposed
. Then, in vivotoevaluation
Aβ25-35 . Then, in vivo evaluation
of compound 105 using the of Morrison
compound
105 using the Morrison Water Maze method [162] was
Water Maze method [162] was made and valuable results based on memory improvement made and valuable results basedinon
memory improvement
scopolamine-induced in scopolamine-induced
impairment were observed. Similarly, impairment were and
Rastegari observed. Similarly,
collaborators Rastegari
synthesized
and collaborators
a series synthesized a series
of 1,2,3-triazole-chromenone of 1,2,3-triazole-chromenone
carboxamide derivatives and investigated carboxamide derivatives
their potential and
as anti-
investigated their potential as anti-Alzheimer’s agents, in terms of inhibitory
Alzheimer’s agents, in terms of inhibitory activity against AChE, BuChE and BACE1, besides their activity against AChE,
BuChE and BACE1,
neuroprotective and besides their neuroprotective
metal-chelating properties and [163].metal-chelating
The anti-AChE properties [163].
activity of The anti-AChE
coumarin-3-
activity of coumarin-3-carboxamide
carboxamide was already known [164], was asalready
well asknown [164],
the ability ofas well
the as the ability
1,2,3-triazole of theto
moiety 1,2,3-triazole
enhance
moiety
AChEI to enhance
activity, AChEI
if linked withactivity,
tacrineiforlinked with tacrine orderivatives
phenanthridinium phenanthridinium derivatives
and its BACE1 and its
inhibitory
BACE1[165].
potential inhibitory
In vitro potential
AChEI and [165].
BuChEIIn vitro AChEI
activities of and BuChEI
the new activities
compounds of the
were new compounds
evaluated, keeping
were evaluated, keeping donepezil as reference. Higher activities
donepezil as reference. Higher activities were shown by compound 107, bearing 3,4-dimethylbenzyl were shown by compound
107, bearing
connected 3,4-dimethylbenzyl
to 1,2,3-triazole moietyconnected
and by compoundto 1,2,3-triazole moiety and
108, having by compound 108,
3-morpholinopropyl andhaving
2-
3-morpholinopropyl
bromobenzyl and 2-bromobenzyl
moieties (Figure 41), even if they moieties (Figure
are much less41), even
active thanif they are much
donepezil less
(IC50 active
=0.027 μM).than
Anti-BChE (IC50 =was
donepezilactivity 0.027
alsoµM). Anti-BChE
modest activity
and resulted to was also modest
be affected by theandtyperesulted
of amine toconnected
be affectedtoby thethe
type moiety,
amide of aminethatconnected to the amide
is, morpholine moiety, that
or piperidine andis,bymorpholine
the position or piperidine
and electronicand properties
by the position
of
and electronic
substituents on theproperties of substituents
benzyl group connectedon tothe benzyl group
1,2,3-triazole ring.connected to 1,2,3-triazole ring.
Figure 41. Chemical structure and IC50 values of compounds 107 and 108.
Figure 41. Chemical structure and IC50 values of compounds 107 and 108.
Compound 107 was chosen for BACE1 inhibitory activity evaluation, showing only modest
results (IC50 =107
Compound 21.13
wasµM compared
chosen to the
for BACE1 referenceactivity
inhibitory OM99-2 having IC50
evaluation, = 0.014
showing onlyµM). Also,
modest
neuroprotective
results potential
(IC50 = 21.13 of 107 wastoinvestigated
μM compared the reference by comparing
OM99-2 having IC50 =cells
107-treated with intact
0.014μM). Also,(no
intervention), quercetin+H O
neuroprotective potential of2 107 -treated (positive control) and
2 was investigated by comparing H O -treated (negative control) cells.
2 2 107-treated cells with intact (no The
mentioned compound showed moderate to good neuroprotective activity, not stronger than
intervention), quercetin+H2O2-treated (positive control) and H2O2-treated (negative control) cells. The quercetin.
Finally, since ROS, which are potentially involved in the neurodegenerative process of AD, may be
mentioned compound showed moderate to good neuroprotective activity, not stronger than
generated from unregulated reaction of molecular oxygen with the redox active metals such as Fe, Cu
quercetin. Finally, since ROS, which are potentially involved2+, in the neurodegenerative process of AD,
and Zn, the metal-chelating properties of 107 toward Cu Fe2+ and Zn2+ were tested in methanol
may be generated from unregulated reaction of molecular oxygen with the redox active metals such
as Fe, Cu and Zn, the metal-chelating properties of 107 toward Cu2+, Fe2+ and Zn2+ were tested in
methanol by means of UV-vis spectrophotometry. Interaction between 107 and Cu2+ and Zn2+ was
detected, whereas no interaction was observed between 107 and Fe2+.
Int. J. Mol. Sci. 2020, 21, 4618 26 of 81

by means of UV-vis spectrophotometry. Interaction between 107 and Cu2+ and Zn2+ was detected,
whereas no interaction
De Souza was observed
and co-workers between
designed and 107 and Fe2+ .a series of 3-substituted-7-aminoalcoxy-
synthesized
De Souza
coumarin and co-workers
derivatives (109a–d, designed and synthesized
110a–c, 111a–c, Figure 42)a series
whoseofAChEI/BuChEI
3-substituted-7-aminoalcoxy-
activities and
coumarin
antioxidant properties were evaluated [166]. All the compounds showed good inhibitory activityand
derivatives (109a–d, 110a–c, 111a–c, Figure 42) whose AChEI/BuChEI activities on
antioxidant
AChE, with properties
potencies inwere evaluated [166].
the nanomolar range, All the compounds
whereas showed
their activity againstgood
BuChEinhibitory
was loweractivity
(IC50
on AChE, 0.90
between with to
potencies in the 3-(4-(dimethylamino)phenyl)-7-aminoethoxycoumarin
15.85 μM); nanomolar range, whereas their activity against BuChE(111a) was lowerwas
(IC between
considered
50 0.90 to 15.85 µM); 3-(4-(dimethylamino)phenyl)-7-aminoethoxycoumarin
a hit, showing an excellent inhibitory activity (IC50 = 20 nM) and selectivity towards AChE (111a) was
considered a hit, showing
(IC50 BuChE/AChE an excellenttoinhibitory
= 354) compared activity
the reference drug(IC 50 = 20 nM)
donepezil (ICand
50 = 6selectivity towards AChE=
nM, IC50 BuChE/AChE
(IC50 BuChE/AChE
365). Investigation of = 354) compared
antioxidant to the reference
properties showed drug
thatdonepezil (IC50 = 6111a–c
only compounds nM, ICpresented
50 BuChE/AChEactivity=
365). Investigation of antioxidant properties showed that only compounds 111a–c presented
in Ferric Ion Reduction Method (FRAP), whereas series 109a–d and 110a–c did not show significant activity in
Ferric
results,Ionsuggesting
Reduction that
Method
the (FRAP), whereas series 109a–d
dimethylaminophenyl moietyandmay110a–c did not show
be responsible forsignificant results,
the antioxidant
suggesting
activity. that the dimethylaminophenyl moiety may be responsible for the antioxidant activity.
Figure 42. Chemical

Figure 42. Chemical structures
structures of
of alkylamino
alkylamino coumarins
coumarins 109a–d,
109a–d, 110a–c and 111a–c.
110a-c and 111a–c.
Docking
Docking simulations
simulations showed
showed that all the
that all the compounds
compounds were were able
able to
to bind
bind simultaneously
simultaneously the the PAS
PAS
and cationic active site (CAS) of the electric eel AChE (eeAChE): the interaction
and cationic active site (CAS) of the electric eel AChE (eeAChE): the interaction with the CAS took with the CAS took
place
place by
by means
means of of cation-π
cation-π interaction
interaction between
between piperidinyl
piperidinyl groupgroup andand Trp86.
Trp86. Compounds
Compounds 111a 111a and
and
111c showed the ability to give a π-stacking interaction in the PAS, with Trp286.
111c showed the ability to give a π-stacking interaction in the PAS, with Trp286. The smaller spacer The smaller spacer of
111a allowed the coumarin moiety to be located properly in the gorge, achieving
of 111a allowed the coumarin moiety to be located properly in the gorge, achieving H-bonds with H-bonds with Tyr337
and Phe295.
Tyr337 This is This
and Phe295. probably the reason
is probably for thefor
the reason most efficient
the most binding
efficient mode
binding (and(and
mode consequently
consequently the
best activity) to eeAChE of compounds with a short
the best activity) to eeAChE of compounds with a short spacer. spacer.
Yang
Yang and
and his
his group
group synthesized
synthesized aa series
series ofof twenty-four
twenty-four 3-aryl
3-aryl coumarin
derivatives and
and tested
tested
their cholinesterase inhibitory activity, monoamine oxidase (MAO) inhibitory
their cholinesterase inhibitory activity, monoamine oxidase (MAO) inhibitory activity and activity and antioxidant
activity in vitro
antioxidant [167].
activity As far[167].
in vitro as cholinesterase inhibition isinhibition
As far as cholinesterase concerned, is most of themost
concerned, mentioned
of the
compounds showed moderate activity. Compound 117 (Figure 43)
mentioned compounds showed moderate activity. Compound 117 (Figure 43) exhibited a strong exhibited a strong activity (IC50
= 3.04 ±(IC
activity 0.32 µM), whereas compound 116 showed selectivity towards AChE. Compound 114 was
50 = 3.04 ± 0.32 μM), whereas compound 116 showed selectivity towards AChE. Compound
more
114 was more activeBuChE
active against against(IC 50 = 2.76
BuChE (IC± 0.57 µM) than donepezil, whereas compounds 112, 113 and
50 = 2.76 ± 0.57 μM) than donepezil, whereas compounds 112,
113 showed
115 and 115 selectivity towards towards
showed selectivity BuChE. 3-Arylcoumarins
BuChE. 3-Arylcoumarins bearing bearing
hydroxyhydroxy
group both
groupat R 5 and
both at RR65
positions displayed higher activity respect to the mono-substituted counterparts,
and R6 positions displayed higher activity respect to the mono-substituted counterparts, especially especially towards
AChE,
towards whereas
AChE, no significant
whereas impact onimpact
no significant BuChEoninhibition was observed.
BuChE inhibition was observed.
Figure 43. General structure of compounds 112–117.

antioxidant activity in vitro [167]. As far as cholinesterase inhibition is concerned, most of the
mentioned compounds showed moderate activity. Compound 117 (Figure 43) exhibited a strong
activity (IC50 = 3.04 ± 0.32 μM), whereas compound 116 showed selectivity towards AChE. Compound
114 was more active against BuChE (IC50 = 2.76 ± 0.57 μM) than donepezil, whereas compounds 112,
113 and 115 showed selectivity towards BuChE. 3-Arylcoumarins bearing hydroxy group both at R5
Int. J. Mol. Sci. 2020, 21, 4618 27 of 81
and R6 positions displayed higher activity respect to the mono-substituted counterparts, especially
towards AChE, whereas no significant impact on BuChE inhibition was observed.
Figure 43. General

General structure
structure of compounds 112–117.
As mentioned, MAO-inhibitory activity was evaluated as well. In fact, MAO is one of the
enzymes responsible for oxidative stress, which is another factor involved in the neurodegenerative
process characterizing AD and different studies identified various ChE/MAO inhibitors as tools for AD
treatment, showing positive results in clinical trials [168]. In Yang’s work, 3-arylcoumarins anti-MAO
activity was compared to that of rasagiline (a selective MAO-B inhibitor): almost half of the compounds
showed relevant activity [167]. Among them, compound 117 was the most promising, with an IC50
= 27.03 ± 0.50 µM, though weaker than rasagiline (IC50 = 0.125 ± 0.005 µM). Again, the presence of
R5 , R6 hydroxy groups provided better MAO inhibitory activity. Finally, the antioxidant power of
3-arylcoumarins was studied by means of Ferric Ion Reduction Method (FRAP), using vitamin C as a
reference. Compound 117 resulted again the most active (FRAP value = 41.42 ± 0.35), also showing a
certain activity in vivo, when tested on zebrafish, leading to an increase of total distance of zebrafish
movement proportional to the concentration of compound used.
In 2017, Joubert and collaborators designed and synthesized a series of 7-substituted coumarins
(118–146, Figure 44), consisting in a coumarin motif (MAO inhibitor) condensed with a benzyl-,
piperidinyl-, N-benzylpiperidinyl- or p-bromo-N-benzylpiperizinyl moiety, which resemble the
N-benzylpiperidine function present in donepezil (AChE inhibitor), connected via an alkyl ether
linkage at position 7 [169]. Their biological activities were evaluated in terms of MAO and ChE
inhibitory potential. Inhibition of hMAO was achieved from all the designed compounds, which
also showed selectivity towards MAO-B. The benzyloxy series showed higher activity, in some cases
even better than the reference selegiline (IC50 = 0.008 µM), with IC50 values in the nanomolar range
(0.5–73 nM).
piperidinyl-, N-benzylpiperidinyl- or p-bromo-N-benzylpiperizinyl moiety, which resemble the N-
benzylpiperidine function present in donepezil (AChE inhibitor), connected via an alkyl ether linkage
at position 7 [169]. Their biological activities were evaluated in terms of MAO and ChE inhibitory
potential. Inhibition of hMAO was achieved from all the designed compounds, which also showed
selectivity towards
Int. J. Mol. Sci. 2020,MAO-B.
21, 4618 The benzyloxy series showed higher activity, in some cases even better
28 of 81
than the reference selegiline (IC50 = 0.008 μM), with IC50 values in the nanomolar range (0.5-73 nM).
Figure
Figure 44. (a) (a) General
44.General structure
structure of compounds
of compounds 118–146;
118–146; (b) chemical
(b) chemical structure
structure of compounds
of compounds 120 and 120
136. and 136.
The presence of piperidine or piperazine ring caused a decrease in MAO inhibitory activity,
The presence of piperidine or piperazine ring caused a decrease in MAO inhibitory activity,
perhaps indicating that larger substitutions at position 7 are not well tolerated. Again, no activity
perhaps indicating that larger substitutions at position 7 are not well tolerated. Again, no activity
against MAO-A was shown, whereas IC50 values between 0.29 and 5.64 µM were detected on
against MAO-A was shown, whereas IC50 values between 0.29 and 5.64 μM were detected on MAO-
MAO-B. Compounds that lacked the N-benzylpiperidine, responsible for anti-cholinesterase activity
B. Compounds that lacked the N-benzylpiperidine, responsible for anti-cholinesterase activity in
in donepezil, showed little or no activity against eeAChE and equine BuChE (eqBuChE). Compounds
donepezil, showed little or no activity against eeAChE and equine BuChE (eqBuChE). Compounds
bearing the N -benzylpiperidin moiety showed moderate inhibitory activity against both ChEs (IC
bearing the N -benzylpiperidin moiety showed moderate inhibitory activity against both ChEs (IC50 50
values between 1.42 and 10.22 µM) with some slight selectivity towards BuChE. Compounds 120 and
values between 1.42 and 10.22 μM) with some slight selectivity towards BuChE. Compounds 120 and
136 (Figure 44b) resulted the most promising. In fact, molecular modelling revealed that compound
136 (Figure 44b) resulted the most promising. In fact, molecular modelling revealed that compound
120 can realize favorable interactions with the active sites of both BuChE and MAO-B. This double
120 can realize favorable interactions with the active sites of both BuChE and MAO-B. This double
activity may be advantageous in AD treatment, because both enzymes are becoming relevant biological
activity may be advantageous in AD treatment, because both enzymes are becoming relevant
targets for the treatment of this dementia. Also compound 136 resulted to be a potential MTDL
biological targets for the treatment of this dementia. Also compound 136 resulted to be a potential
because it showed inhibitory activity against hMAO-B (IC50 = 0.30 µM), eeAChE (IC50 = 9.10 µM)
MTDL because it showed inhibitory activity against hMAO-B (IC 50 = 0.30 μM), eeAChE (IC50 = 9.10
and eqBuChE (IC50 = 5.90 µM). Docking studies showed that 136 binds the substrate cavity through
μM) and eqBuChE (IC 50 = 5.90 μM). Docking studies showed that 136 binds the substrate cavity
the coumarin ring, whereas the benzyl moiety occupies the entrance cavity. In addition, 136 was
through the coumarin ring, whereas the benzyl moiety occupies the entrance cavity. In addition, 136
able to bind both the CAS and PAS of cholinesterase, which suggested that it could interfere with
PAS-induced β-amyloid aggregation. A different approach was followed by Shi and collaborators,
who designed four derivatives obtained by coupling two pharmacophores (carbazole and coumarin)
to obtain potential multitarget agents for the treatment of AD [170]. The aim was to exploit the
biological activities of both the mentioned moieties: on one hand coumarins are known to have
inhibitory activity on AChE, BuChE and MAO, besides antioxidant properties; on the other hand,
carbazole exhibits antioxidant activity [171] and the ability to inhibit Aβ aggregation [172]. In addition,
carbazole derivatives are reported to have inhibitory effect on ChEs, thus providing neuroprotection
from β-amyloid toxicity [173]. Thus, compounds 147a–d (Figure 45) were synthesized, characterized
and their biological activities were evaluated: anticholinesterase activity was tested on eeAChE and
on hAChE, using Ellman’s method [159], keeping tacrine as reference drug. Whereas all derivatives
showed dose-dependent inhibitory activity on eeAChE (compound 147d resulted the best with an IC50
of 3.75 µM), on hAChE they exhibited weak effects (147b IC50 = 76.63 µM, 147d IC50 = 70.51 µM). None
of the tested compounds inhibited eqBuChE or hBuChE more than 30%, which means that compounds
147b and 147d showed selectivity for AChE over BuChE.
synthesized, characterized and their biological activities were evaluated: anticholinesterase activity
was tested on eeAChE and on hAChE, using Ellman’s method [159], keeping tacrine as reference drug.
Whereas all derivatives showed dose-dependent inhibitory activity on eeAChE (compound 147d
resulted the best with an IC50 of 3.75 μM), on hAChE they exhibited weak effects (147b IC50 = 76.63
μM,J. Mol.
Int. 147dSci.
IC2020,
50 = 70.51
21, 4618μM). None of the tested compounds inhibited eqBuChE or hBuChE more 29than
of 81
30%, which means that compounds 147b and 147d showed selectivity for AChE over BuChE.
Figure 45. Chemical

structure of
of compounds
compounds 147a–d.
147a–d.
As
As the
the alkyl
alkyl linker
linker length
lengthincreased,
increased,thetheanti-cholinesterase
anti-cholinesteraseactivity
activityimproved,
improved,probably because
probably because a
5-methylene chain allowed the carbazole and coumarin moieties to bind both the CAS
a 5-methylene chain allowed the carbazole and coumarin moieties to bind both the CAS and the PAS, and the PAS,
respectively.
respectively. Antioxidant
Antioxidant properties
properties were
were evaluated
evaluated through
through the
the ORAC-FL
ORAC-FL method
method (oxygen
(oxygen radical
radical
absorbance capacity by fluorescein) [174], using Trolox (vitamin E analogue) as a
absorbance capacity by fluorescein) [174], using Trolox (vitamin E analogue) as a standard; standard; none of the
none of
tested compounds showed significant activity. Among the tested derivatives, compound
the tested compounds showed significant activity. Among the tested derivatives, compound 147d 147d resulted
aresulted
promising lead candidate
a promising for the treatment
lead candidate of AD. of AD.
for the treatment
2.9. Anticonvulsant Activity
2.9. Anticonvulsant Activity
Epilepsy is a widespread neurological disorder, characterized by periodic and unpredictable
Epilepsy is a widespread neurological disorder, characterized by periodic and unpredictable
attacks, involving seizures and/or transient behavioral changes. Its pathogenesis has not been
attacks, involving seizures and/or transient behavioral changes. Its pathogenesis has not been
completely clarified yet; it is known, however, that an impairment between excitatory and inhibitory
completely clarified yet; it is known, however, that an impairment between excitatory and inhibitory
neurotransmission is involved [175–179] Here, we report some recent advances in the use of coumarins
neurotransmission is involved [175–179] Here, we report some recent advances in the use of
as anticonvulsant compounds. Abd-Allah and collaborators recently studied the anticonvulsant activity
coumarins as anticonvulsant compounds. Abd-Allah and collaborators recently studied the
of a series of coumarin derivatives, achieved by merging two or more pharmacophoric scaffolds in
anticonvulsant activity of a series of coumarin derivatives, achieved by merging two or more
order to create new chemical entities with an improved biological activity [180]. The compounds here
pharmacophoric scaffolds in order to create new chemical entities with an improved biological
described possess all the necessary elements to exert anti-convulsant activity: a lipophilic aryl ring, a
activity [180]. The compounds here described possess all the necessary elements to exert anti-
hydrogen-bonding domain and an electron-donor moiety [178,179,181,182]. All the compounds were
convulsant activity: a lipophilic aryl ring, a hydrogen-bonding domain and an electron-donor moiety
initially screened (phase I) using two standard animal seizure models, subcutaneous pentylenetetrazole
[178,179,181,182]. All the compounds were initially screened (phase I) using two standard animal
(scPTZ) and maximal electric shock (MES) seizure tests using ethosuximide as reference drug. An
seizure models, subcutaneous pentylenetetrazole (scPTZ) and maximal electric shock (MES) seizure
assessment of the potential neurotoxicity was also done by means of rotarod test. Phase II consisted
tests using ethosuximide as reference drug. An assessment of the potential neurotoxicity was also
in the determination of ED50 value for compounds that conferred 100% protection in one or both
done by means of rotarod test. Phase II consisted in the determination of ED50 value for compounds
tests. In the end, GABA level measurements were carried out in whole mouse brain for the most
that conferred 100% protection in one or both tests. In the end, GABA level measurements were
active compounds, using gabapentin as a reference drug. The results of phase I tests showed that
carried out in whole mouse brain for the most active compounds, using gabapentin as a reference
all the tested compounds had protective activity against scPTZ-induced absence epilepsy (variable
drug. The results of phase I tests showed that all the tested compounds had protective activity against
results in the range of 17–100% protection). Among them, 148, 149 and 150 (Figure 46) were the most
scPTZ-induced absence epilepsy (variable results in the range of 17-100% protection). Among them,
active (100% protection) at 0.238, 0.239 and 0.283 mmol/kg, meaning that the compounds are 1.49, 1.48,
148, 149 and 150 (Figure 46) were the most active (100% protection) at 0.238, 0.239 and 0.283 mmol/kg,
1.25 folds more potent than ethosuximide, respectively. In the MES-induced seizures though, all the
meaning that the compounds are 1.49, 1.48, 1.25 folds more potent than ethosuximide, respectively.
compounds failed in completely protecting the animals. The best profile was exhibited by compound
151 (Figure 46) which was capable of a 50% protection at 2.1 mmol/kg. A quantitative determination
of ED50 values was carried out for compounds 148, 149 and 150, which were able to fully protect
animals in the scPTZ test. Compound 148 was found to be the most active with an ED50 of 54.86 mg/kg
(0.131 mmol/kg). Consequently, it was chosen for further investigation to elucidate the mechanism
of action, which was assessed through an evaluation of GABA levels in mice brain. As a result, the
proposed mechanism for compound 148 is a GABA-mediated one, perhaps non-vesicular release of
GABA, GABAA receptor activation or GABAB receptor inhibition; probably an enhanced synthesis or
reduced metabolism of GABA are also involved.
the most active with an ED50 of 54.86 mg/kg (0.131 mmol/kg). Consequently, it was chosen for further
investigation to elucidate the mechanism of action, which was assessed through an evaluation of
GABA levels in mice brain. As a result, the proposed mechanism for compound 148 is a GABA-
mediated one, perhaps non-vesicular release of GABA, GABAA receptor activation or GABAB
receptor inhibition;
Int. J. Mol. Sci. 2020, 21,probably
4618 an enhanced synthesis or reduced metabolism of GABA are also
30 of 81
involved.
Figure 46. Chemical structure of compounds 148–151.

A similar bivalent drug approach was followed by Mohammadi-Khanaposhtani and collaborators,
A similar bivalent
who synthesized a seriesdrug approach was followed
of coumarin-1,2,4-oxadiazole by Mohammadi-Khanaposhtani
derivatives in order to create a new chemical and
collaborators, who synthesized
entity with better anticonvulsanta series of coumarin-1,2,4-oxadiazole
profile than coumarin and oxadiazole derivatives in order
alone [183]. to create
In fact, a
different
new chemical entity with better anticonvulsant profile than coumarin and oxadiazole
5-member heterocyclic rings-containing compounds such as oxadiazoles, triazoles and thiadiazoles were alone [183]. In
fact, different
reported 5-member
to have good heterocyclic
anticonvulsant rings-containing compounds
activity [184–186] such as oxadiazoles,
through benzodiazepine (BDZ)triazoles
receptorand [187].
thiadiazoles
The activitywere
of the reported to have good
new derivatives anticonvulsant
was tested using PTZ- activity
and MES-[184–186]
induced through
seizuresbenzodiazepine
in mice, keeping
(BDZ) receptor
diazepam as a[187]. The drug.
reference activity of the
Most new
of the newderivatives
compounds was tested
did usingactivity
not show PTZ- and MES-
against induced
PTZ-induced
seizures
seizures, except for three of them, 152a, 152b and 152c (Figure 47), being 152b the most active show
in mice, keeping diazepam as a reference drug. Most of the new compounds did not (25% of
activity against
protection PTZ-induced
using 10 mg/kg).seizures,
Compounds except152d,
for three
152eofand
them,
152f152a, 152b47)
(Figure and 152c (Figure
showed a 100%47), being
protection
152b the most
against active (25%
MES-induced of protection
seizures using
at the doses 1040
of 7, mg/kg).
and 20 Compounds 152d, 152e
mg/kg, respectively and 152f
(it should (Figure
be considered
47)that
showed a 100%
diazepam showsprotection against MES-induced
100% protection at 2 mg/mL) seizures at the doses
[188]. Compound 152d,of which
7, 40 and 20 mg/kg,
showed the best
respectively (it should be considered that diazepam shows 100% protection at
activity, was characterized by the absence of substituents on position 4 of the coumarin ring and by a 2 mg/mL) [188].
Compound
4-chloroaryl152d,
group which showed
connected tothe
the best activity, was ring.
1, 2, 4-oxadiazole characterized
The most by the compounds
active absence of substituents
152d and 152e
onwere
position
used4toofinvestigate
the coumarin the ring and by of
mechanism a 4-chloroaryl
action; to dogroup
so, theconnected to the 1, 2,(a4-oxadiazole
effect of flumazenil BDZ receptor
ring.
antagonist) on their activity was evaluated. Flumazenil antagonized both 152d and 152e,of
The most active compounds 152d and 152e were used to investigate the mechanism action;
confirming
to that
do so, the effect of flumazenil (a BDZ receptor antagonist) on their activity
these compounds act as BZS receptor agonists. Finally, in vivo neurotoxicity of compounds 152d was evaluated.
Flumazenil
and 152e antagonized
was assessedboth and 152d and 152e,
the tested confirming
compounds gavethatlessthese compounds
neurological act as
deficits BZS
that thereceptor
reference
agonists. Finally,
drug diazepam. in vivo neurotoxicity of compounds 152d and 152e was assessed and the tested
compounds gave less neurological deficits that the reference drug diazepam.
Int. J. Mol. Sci. 2020, 21, 4618 31 of 81
Figure 47. Chemical structure of compounds 152a–f.

Figure
Figure 47. Chemicalstructure
47. Chemical structureof
ofcompounds
compounds152a–f.
152a–f.
A series of 4-amino-3-nitrocoumarins (153a–e, Figure 48) was synthesized and biologically
AA series
evaluated of
of 4-amino-3-nitrocoumarins
by Mokrov
series and co-workers [189]. (153a–e,
4-amino-3-nitrocoumarins (153a–e, Figure 48)
The anticonvulsant
Figure 48) was
wassynthesized
activitysynthesized andbiologically
was investigated
and biologically
using the
evaluated
MES
evaluated by
mice model Mokrov
by Mokrov and
(grandand co-workers
malco-workers
seizures) and [189]. The
theThe
[189]. anticonvulsant
corazole-antagonism
anticonvulsant activity activity was
test (modellinginvestigated
the so-called
was investigated using
using the
petit
the
MES
mal mice
micemodel
MESseizure). The(grand
model mal
mal seizures)
only statistically
(grand seizures) and the
thecorazole-antagonism
significant
and corazole-antagonism
result was given bytest test (modelling
compound
(modelling 153a
thetheso-called
atso-called inpetit
doses petit the
mal
range seizure).
mal seizure). The
of 60-80 Themg/kg,only statistically
onlyasstatistically
it could increasesignificant result
the animal
significant result was was given
survival
giveninbyMESby compound
test up to
compound 153a
153a60% at
at (in doses
dosesthein in the
control
the
range
rangeof
group of60–80
60-80 mg/kg,
survival was 10%).
mg/kg, as
as it could increase the
it Corazole-induced
could increase the animal
animalsurvival
seizures survival ininMES
and animals’ MESdeathtestup
test uptoto60%
were 60%
not (in(in thecontrol
prevented
the controlby
group
groupsurvival
compounds 153a,
survival was153c
was 10%). Corazole-induced
and 153d.
10%). Compound 153e
Corazole-induced seizures
exhibited
seizures andanimals’
and animals’
good deathwere
protection
death werenot
potentialnotprevented
atprevented
10-40 mg/kgbyby
compounds
and was able153a,
compounds 153a, 153cand
153c
to prevent and 153d.
death153d. Compound
in Compound 153e
50-63% of animals. exhibited
153eexhibited
The most good
good protection
protection
active compound potential
potential
in theatat10-40
10–40
MES mg/kg
mg/kg
test was
and
andwas
153a, was able
able to prevent
containing prevent
a coumarin death
death inin50-63%
ring 50–63% of animals.
with aof3-nitro
animals. TheThe
group andmost
most active active compound
compound
a γ-aminobutirric thein
inacid MESthe test
MES
fragment. wastest
The
was 153a,
153a, containing
containing a a coumarin
coumarin ring ring
withwith
a a 3-nitro
3-nitro group
group andanda a γ-aminobutirric
γ-aminobutirric
corresponding methyl-ester (153c) did not show any activity, as well as compound 153d, with an acid
acid fragment.
fragment. TheThe
corresponding
corresponding
additional nitromethyl-ester
methyl-ester
group. Compound (153c) 153e,
(153c) did not
did not showthe
show
bearing any
any activity,as
activity,
methyl-ester aswell
well
of asascompound
GABA compound
pharmacophore 153d,with
153d, withan
and an
two
additional
additional
nitro groups nitro
nitro group.
ongroup. Compound
Compound
the coumarin 153e,
153e,
ring, bearing
bearing
showed thethe
the bestmethyl-ester
methyl-ester
profile ofinGABAof GABA pharmacophore
pharmacophore
corazole-antagonism and
test.twoand
The
nitro
two groups
nitro groups on the
on coumarin
the coumarin ring,
ring,showed
showed the the best
best profile
profile inin
corresponding free carboxylic acid 153d was found to be inactive, as well as compound 153c, having corazole-antagonism
corazole-antagonism test.
test.TheThe
corresponding
corresponding
one less nitro group.freecarboxylic
free carboxylic
Compound acid 153d
153a, was
was found
bearing found to
one nitrotobe
beinactive,
inactive,
group andas well
asnowell asascompound
compound
substituent on GABA153c,
153c,having
having
moiety,
onenot
one
did lessshow
less nitrogroup.
nitro group.
any Compound 153a,
corazole-antagonist bearing
bearingone
153a,activity. onenitro
nitrogroup
Therefore, group
it canand beno
and nosubstituent
substituent
speculated thatonon GABAGABA
compounds moiety,
moiety,
153a
did
did not
not show
show any
any corazole-antagonist
corazole-antagonist activity.
activity. Therefore,
Therefore, it
itcan
can bebe
and 153e possess different mechanisms responsible for their anticonvulsant activity and they may speculated
speculated that
that compounds
compounds 153a
153a
be
and
aand 153e possess
153e possess
starting point for different
different mechanisms
furthermechanisms responsible
responsible
studies on coumarin for their anticonvulsant
for their anticonvulsant
derivatives activity
as anticonvulsant agents. and they may be
be a
a starting
starting point
point forfor further
further studies
studies onon coumarinderivatives
coumarin derivativesasasanticonvulsant
anticonvulsantagents. agents.
Figure 48. Chemical structure of compounds 153a–e.

2.10. Anticoagulant Activity
2.10. Anticoagulant
2.10.The Activity
recent medical
Anticoagulant history of anticoagulant drugs has been largely dominated by a simple class of
Activity
molecules
The that has been discovered thanks to a mysterious livestock mortality and, later, employed as a
Therecent
recentmedical
medical history
history of
of anticoagulant drugshas
anticoagulant drugs has beenlargely
been largely dominated
dominated bybya asimple
simple class
class 0
powerful rodenticide:
ofofmolecules coumarins. The anticoagulant activity of coumarins was identified when employeds
in 1920
moleculesthat
thathas
hasbeen
been discovered thanks to
discovered thanks to aa mysterious
mysteriouslivestock
livestockmortality
mortalityand, and,later,
later,
employed
seemingly
as healthy cattle of Canada and North America diedactivityinexplicably for internal hemorrhages. The
asaapowerful
powerfulrodenticide:
rodenticide: coumarins.
coumarins. TheThe anticoagulant
anticoagulant activity ofofcoumarins
coumarins was
was identifiedwhen
identified when
main
in causeseemingly
in1920′s
1920′s of this decimation
seemingly healthy was
healthy attributed
cattle
cattle of Canada
of to aand
Canada mold
and infestation
North
North of the
America
America damp
died
died hay, later for
inexplicably
inexplicably known as the
forinternal
internal
“sweet clover
hemorrhages.
hemorrhages.The disease.”
The main However,
main cause
cause of it was
of this not before
decimation was
this decimation 1940 that
was attributedthe responsible
attributedtotoaamold molecule
moldinfestation was
infestationofofthe identified
thedamp
damp
by
hay,Karl
hay, Link
later
later and as
known
known his student
as the
the “sweet
“sweetHarold
cloverCampbell: it was 3,3’-methylenebis(4-hydroxycoumarin),
disease.” However,
disease.” However, ititwas
wasnot
notbefore
before19401940that
thatthe later
theresponsible
responsible
known
molecule
molecule aswas
dicoumarol.
identifiedFurther
wasidentified by Karlstudies
by Karl Link and by his
Link’s
his teamHarold
student
student brought
Harold to the discovery
Campbell:
Campbell: ititwas of warfarin in 1948
was3,3’-methylenebis(4-
3,3’-methylenebis(4-
hydroxycoumarin), later known as dicoumarol. Further studies by Link’s team brought to the
discovery of warfarin later
hydroxycoumarin), in 1948 (so named
known from WARF,
as dicoumarol. Wisconsin
Further Alumni
studies Research
by Link’s teamFoundation,
brought tothatthe
Int. J. Mol. Sci. 2020, 21, 4618 32 of 81
financed
discovery theofproject),
warfarinapproved
in 1948 (so as named
a rodenticide in the USA
from WARF, in 1952Alumni
Wisconsin and forResearch
anticoagulation therapy
Foundation, that
infinanced
human in the1954, under
project), the nameasofa Coumadin.
approved rodenticideCurrently,
in the USAwarfarin is one
in 1952 and forofanticoagulation
the most widelytherapy
used
anticoagulation
in human
(so named in from drug
1954, (in
under
WARF, the UK it has
the nameAlumni
Wisconsin been estimated
of Coumadin.
Research that
Currently, at least
warfarin
Foundation, 1% of the population
is one ofthe
that financed theproject),and
most widely 8% of
used
approved
people
as over 80 years
anticoagulation
a rodenticide in theare
drug (intaking
USA the
in UKwarfarin),
1952 itand
has for together
been withthat
estimated
anticoagulationother at coumarin
leastin1%
therapy derivatives
of
human 1954, like
theinpopulation
underdicumarol
and
the 8%
name of
and acenocoumarol
people over 80 years(Figure
are 49)
taking[18,190–192].
warfarin), together with other coumarin derivatives
of Coumadin. Currently, warfarin is one of the most widely used anticoagulation drug (in the UK it has like dicumarol
and acenocoumarol
been estimated that at (Figure
least 1%49)of[18,190–192].
the population and 8% of people over 80 years are taking warfarin),
together with other coumarin derivatives like dicumarol and acenocoumarol (Figure 49) [18,190–192].
Figure 49. Chemical structure of the most common anticoagulant drugs.

Warfarin
Warfarinand andother
otheranticoagulant
anticoagulantcoumarins
coumarinsare arevitamin-K
vitamin-Kantagonists
antagonists(VKAs).
(VKAs).Indeed,Indeed,due duetoto
the
thestructural
Warfarin
structural similarity
and other
similarity with vitamin-K,
vitamin-K,the
anticoagulant
with thecompounds
coumarins
compounds block
blockthe
are vitamin-K thevitamin-K
vitamin-Kdependent
antagonists (VKAs). Indeed,
dependent pathways
pathways dueofof
to
coagulation,
the structural involving
similarity several
with factors
vitamin-K, (II, VII,
the IX and
compounds X). Despite
block thethe efficacy
vitamin-K
coagulation, involving several factors (II, VII, IX and X). Despite the efficacy and the advantages of an and the
dependent advantages
pathways ofof
an
oral oral therapy,
coagulation,
therapy, warfarinwarfarin
involving notisdevoid
isseveral not devoid
factors of side
(II, VII,
of side IX and
effects, effects,
mainly mainlythe
X). associated
Despite associated
efficacy
with with
and
bleeding thebleeding
and advantages
complications,andof
complications,
an oral
like therapy,
the narrow like the narrow
warfarin
therapeutic range therapeutic
is notand devoid of range and interindividual
side effects,
interindividual genetic mainly
differenceassociatedgenetic
with difference
in pharmacokinetics, bleeding in
and
which
pharmacokinetics,
complications,
requires a continuous which
like the requires a continuous
narrow oftherapeutic
monitoring monitoring of the patient [193,194].
range and interindividual genetic difference in
the patient [193,194].
For
Forthis
thisreason,
pharmacokinetics, reason, the
theresearch
which requiresofaofnew
research newsafer
continuoussaferand efficient
monitoring
and compounds
of
efficient the patientlead
compounds to to
[193,194].
lead thethediscovery
discovery of aof
novel VKA,
For thistecarfarin
reason, (ATI-5923,
the research Figure
of new 50), currently
safer and under
efficientdevelopment
compounds
a novel VKA, tecarfarin (ATI-5923, Figure 50), currently under development [195]. Tecarfarin [195].
lead toTecarfarin
the is
discovery active
ofisa
after
active oral
novel VKA, administration
after oraltecarfarin and
(ATI-5923,
administration acts as
Figure
and a
acts as vitamin-K
50), epoxide
currently under
a vitamin-K epoxide reductase
development (VKOR) inhibitor;
[195]. Tecarfarin
reductase (VKOR) unlike
inhibitor;isunlike
active
warfarin,
after oralis not metabolized
administration by
and the
actscytochrome
as a P450
vitamin-K system
epoxidebut by human
reductase
warfarin, is not metabolized by the cytochrome P450 system but by human carboxylesterase-2 (hCE-2) carboxylesterase-2
(VKOR) inhibitor; (hCE-
unlike
2)ininhepatic
hepatic
warfarin, microsomes.
ismicrosomes.
not metabolized Consequently, drug-drug
by the cytochrome
Consequently, drug-drug P450or food-drug
or food-drug
system butinteractions are
are avoided,
avoided,as
by human carboxylesterase-2
interactions aswell as
(hCE-
well as
genetic
genetic variability
2) in hepatic of
microsomes.
variability CYP-450
of CYP-450 system,
Consequently, providing
drug-drug
system, providing a more stable
or food-drug
a more anticoagulation effect
interactions are effect
stable anticoagulation compared
avoided, as welltoto
compared as
warfarin
genetic [196].
variabilityA detailed
of study
CYP-450 on pharmacokinetics
system, providing a and
more pharmacodynamics
stable anticoagulation
warfarin [196]. A detailed study on pharmacokinetics and pharmacodynamics of tecarfarin had been of tecarfarin
effect had
compared been to
conducted
warfarin [196].
conducted on healthy
A detailed
on healthy patients
patientsstudy by Albrecht et
on pharmacokinetics
by Albrecht al., together
et al., together and with the
pharmacodynamics
with recent
the recent phase I studyphase I
of tecarfarinstudy on
had been
on its tolerabilityits
tolerability
conducted
among among
patients patients
on healthy
with severe with
patients severe
kidney kidney[197,198].
by Albrecht
disease disease [197,198].
et al., together Tecarfarin
with
Tecarfarin thethe
has has
recent the potentiality
phase
potentiality I to be a to
study on beits
valid
asubstitute
valid substitute
tolerability among of warfarin
patients in
withthe oral
severe therapy
kidney of thromboembolic
disease
of warfarin in the oral therapy of thromboembolic disease. [197,198]. disease.
Tecarfarin has the potentiality to be
a valid substitute of warfarin in the oral therapy of thromboembolic disease.
Figure 50. Structure of Tecarfarin.
Figure 50. Structure of Tecarfarin.

Another approach reported in literature is the chemical modification of the coumarin scaffold
Figure
by conjugation of 7-hydroxylcoumarin 50. 7-hydroxy-4-methylcoumarin
and Structure of Tecarfarin. with some derivatives of
salicylic acid. Among the compounds evaluated by Bang and co-workers in 2019, derivatives 154
and 155 (Figure 51) showed high anticoagulant activity, with an increased prothrombin time (PT) of
Another approach reported in literature is the chemical modification of the coumarin scaffold
by conjugation of 7-hydroxylcoumarin and 7-hydroxy-4-methylcoumarin with some derivatives of
salicylic acid. Among
Another approach thereported
compounds evaluated
in literature is by
theBang and co-workers
chemical modificationin 2019,
of thederivatives 154 and
coumarin scaffold
155 (Figure 51) showed high anticoagulant activity, with an increased prothrombin
by conjugation of 7-hydroxylcoumarin and 7-hydroxy-4-methylcoumarin with some derivatives time (PT) of 10.88
of
Int. J. Mol. Sci. 2020, 21, 4618 33 of 81
salicylic acid. Among the compounds evaluated by Bang and co-workers in 2019, derivatives 154than
± 0.56 sec and 13.10 ± 3.56 sec, respectively. Both compounds resulted 1.5 times more active and
warfarin
155 (Figure (PT 7.97
51) ± 1.93)high
showed [199].
anticoagulant activity, with an increased prothrombin time (PT) of 10.88
± 0.56±sec
10.88 0.56and
s and 13.10
13.10 ± 3.56
± 3.56 sec,s, respectively.
respectively. Both
Both compounds
compounds resulted
resulted 1.5
1.5 times more active than
than
warfarin (PT 7.97
warfarin (PT 7.97 ±± 1.93) [199].
Figure 51. Chemical structure of coumarins conjugated with salicylic acid.
Structural modifications Chemicalof the 4-hydroxycoumarin

structure core was also the rational of Montagut-
Figure
Figure 51.51. Chemical structure ofof coumarins
coumarins conjugated
conjugated with with salicylic
salicylic acid.
acid.
Romans et al., who in 2017 explored the potentiality given by modifications performed on C3 position
Structural
Structuralmodifications
by introducing amodifications
side chain (with ofoftheat
4-hydroxycoumarin
the most one unsaturation)
4-hydroxycoumarin corecore
was also
wasthe
structurally alsorational
related of vitamin-K
to
the rationalMontagut-Romans cofactor
of Montagut-
et al., who
[200].
Romans The in 2017
et underlying
al., who in explored
premise
2017 thewas
explored potentiality
the SAR studygivenperformed
the potentiality by modifications
given by Gebaur
by modifications performed2007, on
inperformed whichC3on position
pointed
C3 position by
out
introducing
that the a
activity side
ofchain (with at
4-hydroxycoumarin most onewasunsaturation)
enhanced structurally
only by related
structural
by introducing a side chain (with at most one unsaturation) structurally related to vitamin-K cofactor to vitamin-K
modification cofactor
for C3 [200].
position
The underlying
by isoprenyl
[200]. The underlying premise
motifs [201]. was
premise the
In this SARthestudy
contest,
was SAR performed
14 functionalized
study by4-hydroxycoumarins
performed Gebaur
by Gebaurin 2007, which
in 2007, withpointed
which alkyl out that
chains
pointed of
out
the activity
different of
length, 4-hydroxycoumarin
both linear and was
branched, enhanced
were only by
synthesized structural
and their
that the activity of 4-hydroxycoumarin was enhanced only by structural modification for C3 position modification
activity was for C3
evaluatedposition
in by
vitro
isoprenyl
andisoprenyl
by ex vivomotifs [201].
(phenprocoumon
motifs [201].InInthis
thiscontest,
was 1414functionalized
included
contest, in the test as4-hydroxycoumarins
functionalized internal standard). The
4-hydroxycoumarins with
with alkyl
alkyl chains
ability to of
inhibit
chains of
different
VKORC1 length,
in rat both
liver linear
microsomes and branched,
was were
evaluated synthesized
in vitro and, and
except their
for
different length, both linear and branched, were synthesized and their activity was evaluated in vitro activity
two was
compounds, evaluated
the in vitro
C3-alkyl
and
and ex
ex vivo
derivatives (phenprocoumon
vivoshowed a sub-micromolar
(phenprocoumon was
was included
activity
included in the
the test
in(from 20 as
test nM
as internal
to 200 standard).
internal nM) The
overcoming
standard). The ability to
to inhibit
the benchmark
ability inhibit
VKORC1
VKORC1 in rat liver microsomes was evaluated in vitro and, except for two compounds, the C3-alkyl
compound in rat liver microsomes
phenprocoumon. was
Further evaluated
ex vivo in vitro
studies and, except
assessed thefor two
ability compounds,
to increase the
in vivo
C3-alkylthe
derivatives
prothrombin
derivatives showed
time (PT)
showed aa sub-micromolar
and compoundsactivity
sub-micromolar 156a and
activity (from
156b20
(from 20 nM
nM to
(Figure 200
to52) nM)
nM) overcoming
200showed a promising
overcoming the
the benchmark
anticoagulant
benchmark
compound
activity after phenprocoumon.
24 h. It is possible Further
that theex vivo
presence studies
of the
compound phenprocoumon. Further ex vivo studies assessed the ability to increase inassessed
halogen the
atom ability
protectsto increase
the drug infrom
vivo the
vivoliver
the
prothrombin
metabolism. time
Despite(PT) theand compounds
interesting 156a and
anticoagulant 156b (Figure
activity, 52)
follow-up
prothrombin time (PT) and compounds 156a and 156b (Figure 52) showed a promising anticoagulant showed
studies a promising
on the liveranticoagulant
metabolism
activity
activity after
after 24
are necessary toh.
24 It is
is possible
determine
h. It that
if these
possible the
the presence
thatmolecules
presence are of the
the halogen
substrate
of atom
atom protects
of CYP2C9,
halogen to which
protects the
theisdrug from
from liver
to attribute
drug the
liver
metabolism.
variability in Despite
the the
dosage interesting
of oral anticoagulant
vitamin-K antagonists,activity,
duefollow-up
to its studies
polymorphism
metabolism. Despite the interesting anticoagulant activity, follow-up studies on the liver metabolism on the liver
[202]. metabolism
are
are necessary
necessary to to determine
determine if if these
these molecules
molecules are are substrate
substrate of of CYP2C9,
CYP2C9, to to which
which is is to
to attribute
attribute the the
variability in the dosage of oral vitamin-K antagonists, due
variability in the dosage of oral vitamin-K antagonists, due to its polymorphism [202].to its polymorphism [202].
OH R 156
a R =4-Cl-Ph
b R =2,4-Cl2-Ph
OH
O R
O 156
a R =4-Cl-Ph
Figure 52. C-3 alkyl coumarin derivatives.
b R =2,4-Cl2-Ph
O O
Concerning nature-derivedFigure coumarins,
52. C-3 endowed withderivatives.
alkyl coumarin promising anticoagulant activity, in 2015
Lei et al. performed a phytochemical investigation on the Chinese herbal medicine Ainsliaea fragrans
earning Concerning nature-derived
five new derivatives. The coumarins,
team evaluatedendowed with promising
the anticoagulant anticoagulant
activity activity,
of all isolates in 2015
via activated
Figure 52. C-3 alkyl coumarin derivatives.
Lei et al. performed a phytochemical investigation on the Chinese herbal
partial thromboplastin time (APTT), thrombin time (TT) and prothrombin time (PT) assays in vitro medicine Ainsliaea fragrans
earning
and in five These
vivo.
Concerning newnature-derived
derivatives. The
studies concluded team
that evaluated
coumarins, one the
withanticoagulant
of the new
endowed compounds activity
(157, Figure
promising anticoagulant of 53)
all presented
isolates
activity, via
in 2015a
activated
remarkable partial thromboplastin
anticoagulant activity time
(PT =(APTT),
41.2s thrombin
and TT 128.5s)time
and(TT)
no and prothrombin
significant
Lei et al. performed a phytochemical investigation on the Chinese herbal medicine Ainsliaea fragranshepatic time
or (PT)
renal assays
toxicity
in vitro five
earning and in vivo.
Int. J. Mol.compared
when 21,to
Sci. 2020,new
These(PT
warfarin
derivatives.
x FOR PEER REVIEW= 55.7s
studies
The concluded
team and that one
TT 80.6s)
evaluated ofAlthough
[203].
the the new further
anticoagulant compounds of (157,
studies
activity allare Figure
necessary
isolates 53)
via
33 of 83
presented
to understand
activated a remarkable
partial mode anticoagulant
thethromboplastin
of actiontime activitythrombin
of compound
(APTT), (PT
157,= it41.2s and
could
time be TT
(TT) 128.5s)
a promising
and and no significant
anticoagulant
prothrombin time (PT) hepatic
agent for
assays
or renal
preclinical toxicity
studies.when compared to warfarin (PT = 55.7s and TT 80.6s) [203].
in vitro and in vivo. These studies concluded that one of the new compounds (157, Figure 53) Although further studies
are necessary
presented to understand
a remarkable the mode
anticoagulant of action
activity of compound
(PT = 41.2s and TT 128.5s)157, and
it could be a promising
no significant hepatic
anticoagulant agent for preclinical studies.
or renal toxicity when compared to warfarin (PT = 55.7s and TT 80.6s) [203]. Although further studies
are necessary to understand the mode of action of compound 157, it could be a promising
anticoagulant agent for preclinical studies.
Figure53.
Figure Coumarinderivative
53.Coumarin derivativefrom Ainsliaeafragrans.
fromAinsliaea fragrans.
2.11. Antidiabetic Activity

Diabetes is a chronic metabolic disease characterized by high blood sugar levels and is generally
caused by an insufficient production of insulin by β-pancreatic cells or by the inability of human body
to use this hormone. The consequences of diabetes might be very serious: blindness, kidney failure,
stroke, heart attacks, lower limb amputation [204–206]
Int. J. Mol. Sci. 2020, 21, 4618 Figure 53. Coumarin derivative from Ainsliaea fragrans. 34 of 81

Diabetes is a chronic metabolic disease characterized by high blood sugar levels and is generally
Diabetes is a chronic production
caused by an insufficient metabolic disease characterized
of insulin by high
by β-pancreatic cellsblood
or bysugar levels and
the inability is generally
of human body
caused by an insufficient production of insulin by β-pancreatic cells or by the inability
to use this hormone. The consequences of diabetes might be very serious: blindness, kidney failure, of human body
to use this
stroke, hormone.
heart attacks, The
lower consequences
limb amputationof diabetes might be very serious: blindness, kidney failure,
[204–206]
stroke, heart attacks, lower limb amputation [204–206]
Menteşe et al. synthesized a novel series of N’-(2-(3,5-disubstituted-4H-1,2,4-triazol-4-yl)acetyl)-
6/7/8Menteşe et al. synthesized a novel series of N0 -(2-(3,5-disubstituted-4H-1,2,4-triazol-4-yl)acetyl)-
substituted-2-oxo-2H-chromen-3-carbohydrazides (158a–e, 159a–e, Figure 54) [207], merging
6/7/8 substituted-2-oxo-2H-chromen-3-carbohydrazides
the 1,2,4-triazole and the coumarin moieties, both characterized (158a–e, 159a–e, by Figure
a wide54)range
[207],ofmerging the
biological
1,2,4-triazole and the coumarin moieties, both characterized by a wide range of
activities (including inhibition of α-glucosidases) and low toxicity profiles [208–213]. Then, their biological activities
(including
activity on inhibition of α-glucosidases)
α-glucosidases was studied, and low toxicity
evaluating profilesinhibition
the enzyme [208–213].in Then, their activity
the presence of pNPGon
α-glucosidases
(p-nitrophenyl-α- was D-glucopyranoside)
studied, evaluating theasenzyme inhibition
a substrate in in
thethebuffer
presence(pHof pNPG (p-nitrophenyl-α-
6.8). Among the new
D-glucopyranoside) as a substrate in the buffer (pH 6.8). Among the
compounds, four molecules showed high inhibition activity, compared to acarbose (IC new compounds, four molecules
50 = 8.85 ± 0.23
showed high inhibition
μg/ml): 158d (IC50 = 4.28activity, compared
± 0.10 μg/ml), 158eto (IC
acarbose = 8.85
(IC 0.02
50 = 0.96 ± 50
± 0.23
μg/ml), 159d 6.75(IC
(IC50 =158d
µg/mL): 50 =μg/ml)
± 0.10 4.28 ±
0.10 µg/mL), 158e (IC = 0.96
and 159e (IC50 = 1.44 ± 0.06 μg/ml).
50 ± 0.02 µg/mL), 159d (IC 50 = 6.75 ± 0.10 µg/mL) and 159e (IC 50 = 1.44 ±
0.06 µg/mL).
Figure 54. General structures of compounds 158a–e and 159a–e.

Figure 54. General structures of compounds 158a–e and 159a–e.
Compounds 158e and 159e resulted the most active, probably because of the metoxy- group at
position 8 of the coumarin
Compounds 158e and ring.
159eDerivatives
resulted the without
most substituents on positions
active, probably because3of andthe5 of the phenyl
metoxy- group ring
at
linked
position to 8the
oftriazole nucleusring.
the coumarin resulted more active
Derivatives than substituents
without compounds bearing a chlorine
on positions 3 andatom
5 of or
thea phenyl
phenyl
moiety
ring linkedon such positions.
to the triazole According to kinetic
nucleus resulted studies,
more activethe tested
than compounds
compounds inhibit
bearing α-glucosidases
a chlorine atom or
in a competitive way. Other studies focused on coumarins-mediated inhibition
a phenyl moiety on such positions. According to kinetic studies, the tested compounds inhibit α- of α-glucosidases
were carried out
glucosidases in abycompetitive
different groups. Hu and
way. Other collaborators
studies focusedsynthesized through microwave
on coumarins-mediated radiation
inhibition of α-
heating a new series of more than forty 3-arylcoumarins which were screened
glucosidases were carried out by different groups. Hu and collaborators synthesized through for antioxidant activity,
α-glucosidases
microwave radiation inhibition andaadvanced
heating new seriesglycation
of more end-products (AGEs) formation
than forty 3-arylcoumarins whichinhibition [214].
were screened
Only eight of theactivity,
for antioxidant synthesized compoundsinhibition
α-glucosidases (160–167, Figure 55) exhibited
and advanced moderate
glycation to high inhibitory
end-products (AGEs)
Int. J. Mol.on
activity Sci.α-glucosidase.
2020, 21, x FOR PEER REVIEW 34 of 83
formation inhibition [214]. Only eight of the synthesized compounds (160–167, Figure 55) exhibited
moderate to high inhibitory activity on α-glucosidase.
Compound R1 R2 R3 R4 R5 R6 R7 R8
R2 160 CH3 H H H H OH H H
R1 R3 161 H H H H H OH H OH
R8
162 OH H H H H OH H H
R7 163 H H OH H H H OH H
R4
164 CH3 H H H H OH H OH
R6 O O 165 H H OH H H OH H OH
R5 166 OCH3 H OH H H OH H OH
167 H OCH 3 OCH3 H H H OH H
Figure
55. Chemical structure of
of compounds
compounds 160–167.
160–167.
Compound
Compound 165 165 was
was the
the most
most promising
promising(IC (IC50 = 1.37 ± 0.67 µM), with a α-glucosidase inhibitory
50 = 1.37 ± 0.67 μM), with a α-glucosidase inhibitory
activity slightly weaker than acarbose (IC
activity slightly weaker than acarbose 50 = 0.050 ±
(IC50 = 0.050 0.003
± 0.003 From From
µM). μM). an extensive SAR study,
an extensive SARit emerged
study, it
emerged that the 7-hydroxy group is important for the inhibition of α-glucosidase. Compounds 160,
163,164, 165 and 166 were then tested in vivo: none of them showed toxicity on mice (LD50 > 5000
mg/kg). The mentioned compounds were also tested on streptozocin-induced diabetic mice.
Compound 166 showed the best profile, exhibiting a strong reduction of glucose blood levels. Oral
daily administration of 166 (30 mg/kg/day) restored glucose blood levels near normal values,
R6 O O 165 H H OH H H OH H OH
R5 166 OCH3 H OH H H OH H OH
167 H OCH 3 OCH3 H H H OH H

Int. J. Mol. Sci. 2020, 21, 4618 35 of 81
Compound 165 was the most promising (IC50 = 1.37 ± 0.67 μM), with a α-glucosidase inhibitory
activity slightly weaker than acarbose (IC50 = 0.050 ± 0.003 μM). From an extensive SAR study, it
emerged that the 7-hydroxy
that the 7-hydroxy group is important
group is important for the inhibition
for the inhibition of α-glucosidase.
of α-glucosidase. Compounds Compounds
160, 163,160,
164,
163,164,
165 and 165
166 and
were166 were
then thenintested
tested vivo: in vivo:
none of none
them of them showed
showed toxicity toxicity
on miceon(LD mice
50 > (LD
500050 > 5000
mg/kg).
mg/kg). The mentioned
The mentioned compounds compounds were on
were also tested also tested on streptozocin-induced
streptozocin-induced diabetic mice.
diabetic mice. Compound 166
Compound 166 showed
showed the best the best profile,
profile, exhibiting a strongexhibiting
reduction aofstrong reduction
glucose of glucose
blood levels. blood
Oral daily levels. Oral
administration
daily
of 166 administration
(30 mg/kg/day) of 166 (30
restored mg/kg/day)
glucose restored
blood levels nearglucose blood levels
normal values, showingnearan normal values,
effect similar to
showing anoral
that of the effect similar toglibenclamide.
antidiabetic that of the oral antidiabetic glibenclamide.
Asgari
Asgari and
and co-workers
co-workers synthesized
synthesized a new series of biscoumarin-1,2,3-triazole
biscoumarin-1,2,3-triazole derivatives
derivatives
(Figure 56)
56)and
andevaluated
evaluatedtheir α-glucosidase
their α-glucosidaseinhibitory potential,
inhibitory using
potential, acarbose
using as a reference
acarbose drug
as a reference
[215].
drug [215].
Figure 56. Chemical structure of biscoumarin-1,2,3-triazole hybrids. Substituents for compound 168c
Figure 56. Chemical structure of biscoumarin-1,2,3-triazole hybrids. Substituents for compound 168c
are reported.
are reported.
Here, again, two active moieties, both characterized by a wide range of biological activities,
Here,
were merged again, two active
together: the moieties, both and
bis-coumarin characterized by a wide
the 1,2,3-triazole range of[216].
moieties biological activities,
All the were
synthesized
merged
compounds together:
showed theexcellent
bis-coumarin and
activities (ICthe 1,2,3-triazole moieties [216]. All the synthesized
50 between 13.0 ± 1.5 and 75.5 ± 7.0 µM) compared to
compounds showed excellent activities (IC
acarbose (IC50 750.0 ± 12.0). Compound 168c, bearing 2-chloro50 between 13.0 ± phenyl
1.5 andmoiety,
75.5 ±resulted
7.0 μM)the compared to
most active.
acarbose (IC 750.0 ± 12.0). Compound 168c, bearing 2-chloro phenyl
The substitution of the chlorine atom with a methyl group or its shift on the C4 position caused a
50 moiety, resulted the most
active.
decrease The insubstitution of the chlorine
activity. Moreover, atom with
the inhibitory a methyl
activity group
seemed to or its shift
depend on the C4 position
importantly caused
on the electron
aproperties
decrease in activity. Moreover, the inhibitory activity seemed to depend importantly
of the substituents. From further kinetic studies it emerged that compound 168c inhibits on the electron
properties
α-glucosidases of theinsubstituents.
a competitiveFrom modefurther
(Ki = 11 kinetic
µM). studies it emerged that compound 168c inhibits
α-glucosidases in a competitive mode (K = 11 μM).
A different therapeutic approach may be the stimulation of insulin secretion. In this perspective,
i
Ahmed A different therapeutic

and collaborators approach
extracted maythe
from beaerial
the stimulation of insulin
parts of Clutia secretion.
lanceolata In this perspective,
(a medicinal plant native
Ahmed and collaborators
to sub-Saharan Africa andextracted fromPeninsula)
the Arabian the aerial twenty-one
parts of Clutia lanceolata including
coumarins, (a medicinal plant native
methyltio- and
to sub-Saharan Africa and the Arabian Peninsula) twenty-one coumarins,
methylsulfinil-coumarins, thirteen of which were reported for the first time [217]. The structures of including methyltio- and
methylsulfinil-coumarins,
these natural compounds were thirteen of which
elucidated were
from reported
2D-NMR andforMSthespectra,
first time [217]. their
whereas The structures of
anti-diabetic
these
activitynatural
Int. J. Mol.was
compounds
tested
Sci. 2020,
werethe
21, measuring
elucidated
x FOR PEER REVIEW
from 2D-NMR
glucose-triggered and
insulin MS spectra,
secretion whereas
of freshly their murine
isolated anti-diabetic
islets.
35 of 83
activity was tested measuring the glucose-triggered insulin secretion of
Compounds 169, 170 and 171 (Figure 57) resulted the most active in stimulating glucose-triggered freshly isolated murine islets.
Compounds
insulin release, 169, 170 and to
compared
compared 171 (Figure 57)Further
glimepiride. resultedstudies
the most active intostimulating
are needed understandglucose-triggered
structure-activity
relationships
relationships in
in order
order to
to develop
develop new
new active
active compounds.
compounds.
Figure 57. Chemical structure of compounds 169, 170 and 171.

Figure 57. Chemical structure of compounds 169, 170 and 171.
3. Other Applications of Coumarin Scaffold
3.1. Coumarins Photoproperties

The applications and properties of coumarin scaffold have remarkably wide boundaries.
Coumarin-based compounds have been exploited in numerous research and industrial sectors, as
active pharmaceutical ingredients, pesticides, fragrances, dyes for several purposes from laser
Int. J. Mol. Sci. 2020, 21, 4618 36 of 81
3. Other Applications of Coumarin Scaffold
3.1. Coumarins Photoproperties

The applications and properties of coumarin scaffold have remarkably wide boundaries.
Coumarin-based compounds have been exploited in numerous research and industrial sectors,
as active pharmaceutical ingredients, pesticides, fragrances, dyes for several purposes from laser
technology to organic photoredox catalysis, cell imaging, photocleavable protecting groups and
fluorescent biological probes [6,218–225]. In the following paragraphs, the most recent applications
associated with the photophysical properties of coumarins have been reviewed.
3.1.1. Coumarins as Photocleavable Protecting Groups (PPGs)

The development of a suitable formulation is a crucial step in order to achieve new functional
therapeutics. The design of novel strategies aimed at selectively release the bioactive in a specific
district at a determinate time to maximize efficacy and reduce off-target adverse effects represents an
extremely active research frontline. So far, various stimuli-responsive systems have been considered
in therapeutic approaches to regulate the release of the therapeutic cargo, including endogenous
stimuli (e.g., pH, enzymes, redox reactions, etc.) and exogenous stimuli (e.g., light, magnetic field,
ionizing radiations, etc.) [226,227]. Light-mediated therapies have shown excellent results in achieving
on-demand therapeutics and optical tools for studying and controlling complex chemical and biological
processes in localized areas, owing to their superior non-invasiveness and spatiotemporal precision
upon applying a specific light-irradiation wavelength [227–229]. One method for the regulation of
molecular processes with light is the use of photolabile “protecting” groups in key locations. Ideally,
this modification completely blocks the activity of any molecule and restores it only with light [230].
Coumarins, particularly 4-hydroxymethyl derivatives, are known to undergo photolysis. Keeping
this concept in mind, several biomolecules of interest have been linked to the coumarin nucleus, mostly
as acyl derivatives. Then, under UV irradiation, the biomolecules can be released in biological systems.
The photophysical parameters of the formed derivatives are determined by different factors as the
mode of fusion, the chemical nature of additional rings and the presence of electron-donating and
electron-withdrawing substituents [12].
Fournier and co-workers in 2013 proposed a series of methyl-coumarins with redshifted absorption.
In particular, three compounds (172–174, Figure 58), were synthetically easily accessible and exhibited
a significant action cross section for uncaging with blue-cyan light, whereas their uncaging ability in
the UV spectral domain remained low in order to avoid their photoactivation when a properly tuned
UV illumination is applied [231].
In the same year, Fournier and co-workers further proved that compound 172 was a good
blue-absorbing caging group, owing to its strongly donating substituent conjugated to the thiocarbonyl
group. Moreover, the research team demonstrated that this particular caging group could be
used in zebrafish embryos in the context of development biology to perform chromatic orthogonal
photoactivation of two biologically active species [232].
In 2017, Gandioso and colleagues reported the development of green/red-absorbing chromophores
based on coumarin scaffolds that could be useful as photocleavable protecting groups [224].
A series of coumarin derivatives in which the carbonyl of the lactone was replaced by a
cyano(4-nitrophenyl)methylene moiety, by condensation of a thiocoumarin precursor with the
corresponding arylacetonitrile derivatives, was synthesized and subsequently refined with the insertion
of electro-withdrawing groups at the phenyl ring, leading to absorption in the green to red region (175,
Figure 58) [224]. The insertion of more than one electro-withdrawing group (such as -NO2 and -CN)
decreased the fluorescence emission, whereas the mononitro-containing coumarin derivatives had a
strong emission in the red region upon excitation with green light, as denoted by their significantly large
Stokes shifts. In order to demonstrate the utility of these new compounds as PPGs, a small collection
of coumarin-based photocages of benzoic acid was prepared. Thanks to photolysis studies with green
58) [224]. The insertion of more than one electro-withdrawing group (such as -NO2 and -CN)
decreased the fluorescence emission, whereas the mononitro-containing coumarin derivatives had a
strong emission in the red region upon excitation with green light, as denoted by their significantly
large Stokes shifts. In order to demonstrate the utility of these new compounds as PPGs, a small
collection of2020,
Int. J. Mol. Sci. coumarin-based
21, 4618 photocages of benzoic acid was prepared. Thanks to photolysis studies
37 of 81
with green light, it was demonstrated that the structure of the coumarin chromophore influenced the
rate of the uncaging process. This observation gave the opportunity to exploit these new coumarin
light, it was demonstrated that the structure of the coumarin chromophore influenced the rate of the
scaffolds as caging groups removable with visible light. On the other hand, Bojtar and colleagues
uncaging process. This observation gave the opportunity to exploit these new coumarin scaffolds as
proposed water soluble red-shifted coumarin caging groups (176–178, Figure 58), activated with
caging groups removable with visible light. On the other hand, Bojtar and colleagues proposed water
green-light [233].
soluble red-shifted coumarin caging groups (176–178, Figure 58), activated with green-light [233].
Figure 58. Structure of several coumarin photocleavable protecting groups.

Figure 58. Structure of several coumarin photocleavable protecting groups.
The optical properties of coumarins as photo-responsive unites could be also applied to polymers
The optical properties of coumarins as photo-responsive unites could be also applied to
that after a photochemical activation rapidly degrade into small molecules. In 2018, Iturmendi and
polymers that after a photochemical activation rapidly degrade into small molecules. In 2018,
co-workers proposed that, through functionalization of polyphosphazenes with a coumarin-caged
Iturmendi and co-workers proposed that, through functionalization of polyphosphazenes with a
amino acid as a pendant group along the backbone, the sensitivity of the polymers to hydrolysis would
coumarin-caged amino acid as a pendant group along the backbone, the sensitivity of the polymers
be accelerated upon irradiation and effectively catalyze its own degradation [234].
to hydrolysis would be accelerated upon irradiation and effectively catalyze its own degradation
[234].
3.1.2. Coumarins as Fluorescent Probes
3.1.2.Coumarins
Coumarinspossess a large electron-rich
as Fluorescent Probes π-π conjugated system with charge transfer properties,
reason why coumarin-based fluorophores are widely used for monitoring a variety of biologically
Coumarins
important speciespossess a large electron-rich
and biochemical π-π conjugated
process in living system with
cells, for example charge transfer
as diagnostic agent for properties,
detection
reason why coumarin-based fluorophores are widely used for monitoring a variety
of biothiols, enzymes, mitochondrial pH values, glucose and ions [3,222,235]. In particular, several of biologically
important species and
coumarin scaffolds havebiochemical
been proposed process
and in living cells,
evaluated for thefordetection
exampleofasions diagnostic agent
in different for
fields,
detection of biothiols, enzymes, mitochondrial pH values, glucose and ions [3,222,235].
from cellular imaging to environmental waters. Gong and co-workers based their work on an easily In particular,
several coumarin
synthesized scaffolds have
coumarin-based been proposed
fluorescent probe and(179,evaluated
Figure 59) forthat
the already
detectionwas
of ions in different
effective in the
fields, from cellular imaging to environmental waters.2+ Gong and co-workers based
detection of glutathione (GHS) in the presence of Cu ions, expanding its potentiality to the detection their work on an
easily synthesized coumarin-based fluorescent probe (179, Figure 59) that already was
of hypochlorite ions with high selectivity and sensitivity. The probe showed a remarkable fluorescent effective in the
detection of glutathione
intensity change (GHS)
in response in the presence
to hypochlorite of moreover,
ions; Cu ions, expanding
2+ its potentiality
this probe could be appliedtotothe detection
detect ClO−
of
in hypochlorite ions with
cells via intracellular high selectivity
fluorescent imagingand sensitivity.
[236,237]. Thethe
Given probe showed of
importance a remarkable
hypochlorite fluorescent
ions both
intensity change in response to hypochlorite ions; moreover, this probe could
in living systems (being one of the biologically most important reactive oxygens species) and be applied to detect
in the
ClO - in cells via
environment intracellular
(owing to its use fluorescent imaging
as disinfectant), [236,237].
in 2019, Givenand
Shangguan thecolleagues
importance of hypochlorite
proposed another
probe for this particular ion based on coumarin dye and malononitrile (180, Figure 59). The fluorescence
response of probe 180 at 459 nm towards hypochlorite ions gradually enhanced with the increase of
ClO− concentrations, resulting in 45-fold fluorescence enhancement. Furthermore, probe 180 exhibited
high accuracy for quantitative measurement of hypochlorite ions in real water samples and it can be
used as a potential chemosensor for the detection of ClO− in chemical environmental and biological
systems [238]. In the same year, Tang and co-workers worked on a coumarin based fluorescent probe
(181, Figure 59) able of rapidly discern hypochlorite and copper(II) ions in water sample and biological
systems. Upon the reaction with ClO− , the fluorescence wavelength of 181 displayed a strong blue shift
along with the naked-eye visible changes from yellow to colorless. Moreover, it exhibited an obvious
because of the presence of the 7-diethylamino group and the 3-substituted lactone ring that are well-
known structural pattern accountable for the fluorescence properties. It was observed a different
reactivity profile depending on the pH levels, probably due to the different reactivity of hypochlorite
ions ascribable to the variation of the dissociation of the salt at different pH values. Afterwards, a
deeply investigation on the possible formation of chlorinated derivatives was conducted: HPLC-
Int. J. Mol. Sci. 2020, 21, 4618 38 of 81
PDA-ESI-MS analyses highlighted the presence of chlorinated derivatives and proved that the
chlorination reaction was responsible for the linear fluorescence decays. The results suggest the
possibility
fluorescence to exploit
quenching these coumarin
behavior to ionic probeswith
copper(II) for the detection analysis
colorimetric and quantitative determination
from yellow to luminousof
hypochlorite
yellow [239]. species in vivo.
Figure 59. Coumarin florescent probes for hypochlorite ion detection.
A noteworthyFigure
research59. Coumarin florescent probes
on the mechanism for hypochlorite
of interaction between ioncoumarin
detection. based ionic probes
and hypochlorite ions had been conducted by Starzak and collaborators [240]. First, the research
teamAconfirmed
coumarinthe fluorescent probe in
linear decrease based on a nitro-3-carboxamide
the fluorescence derivative
emissions together for increase
with the selective incopper
ClO−
(II) ions detection was reported by Bekhradnia and colleagues. Compound 185 (Figure
concentration of three different coumarin derivatives, 182–184 (Figure 59), which were selected because 60) showed
the highest
of the fluorescence
presence intensity in presence
of the 7-diethylamino group andof Cuthe compared
2+ to alactone
3-substituted varietyring
of other common
that are heavy
well-known
and toxic metal
structural patternions (for example
accountable Pb(II),
for the Co(II), Hg(II))
fluorescence and inIt aqueous
properties. solution
was observed at 320 nm
a different [241].
reactivity
Another
profile depending on the pH levels, probably due to the different reactivity of hypochlorite2018,
approach was attempted for the selective detection of copper (II) ions by He et al. in ions
which based the fluorescent probe on a coumarin-Schiff base derivative (186, Figure
ascribable to the variation of the dissociation of the salt at different pH values. Afterwards, a deeply60). This probe
resulted to beon
investigation particularly
the possible selective for Cu
formation
2+ even in the presence of several other ions [242]. Saravana
of chlorinated derivatives was conducted: HPLC-PDA-ESI-MS
analyses highlighted the presence of chlorinated derivatives and proved that the chlorination reaction
was responsible for the linear fluorescence decays. The results suggest the possibility to exploit these
coumarin ionic probes for the detection and quantitative determination of hypochlorite species in vivo.
A coumarin fluorescent probe based on a nitro-3-carboxamide derivative for selective copper (II)
ions detection was reported by Bekhradnia and colleagues. Compound 185 (Figure 60) showed the
highest fluorescence intensity in presence of Cu2+ compared to a variety of other common heavy and
toxic metal ions (for example Pb(II), Co(II), Hg(II)) and in aqueous solution at 320 nm [241]. Another
approach was attempted for the selective detection of copper (II) ions by He et al. in 2018, which
based the fluorescent probe on a coumarin-Schiff base derivative (186, Figure 60). This probe resulted
to be particularly selective for Cu2+ even in the presence of several other ions [242]. Saravana Mani
and colleagues designed in 2019 a coumarin hydrazine-based fluorescent probe for the detection
of copper(II), called BENZEPYR (187, Figure 60), exploiting a reaction of condensation between
2-hydrazino benzothiazole and N,N0 -diethylamino-3-acetyl coumarin [243]. This particular fluorescent
chemosensor could selectively detect Cu2+ among other disturbing metal ions, resulting particularly
specific and highly responsive, with a visible colorimetric change of the solution, which turned from
yellow to wine red. Moreover, the limit of detection (LOD) had been estimated to be 40 nM. BENZEPYR
187 was also tested for the fluorescence bioimaging of Cu2+ ions in HeLa cells using fluorescence
microscopic analysis, resulting suitable for the exploitation as an ion marker in living cells.
but also Al ions and amino acids Lys and Arg [244]. In particular, the detection of Lys and Arg took
place with a colorimetric (from yellow to colorless) and a fluorescent response (from a maximum
absorption at 335 nm to 429 nm). At the same time the probe detected either way the presence of Cu2+
ions but could be used only for the fluorescent sensing of Al3+. A further interesting exploitation of
probe 188 (Figure 60) was the fluorescent and colorimetric identification of Cys, Hcy and GSH when
Int. J. Mol. Sci. 2020, 21, 4618 39 of 81
it was complexed with copper ions.
Figure 60. Coumarin fluorescent probes for copper (II) detection.
Another remarkable case

Figure 60.ofCoumarin
coumarin-based chemosensor
fluorescent had been
probes for copper (II)presented
detection. by Li and co-workers,
who synthesized a multifunctional probe able to selectively detect not only copper (II) ions but also
Al3+ Despite
ions andthe key role
amino acids ofLys
chemical
and Argspecies
[244].like
In copper andthe
particular, hypochlorite,
detection of they
Lysare
andnot
Argthetook
onlyplace
ions
valuable for detection. For this reason, a dual coumarin probe, fluorescent
with a colorimetric (from yellow to colorless) and a fluorescent response (from a maximum absorption and colorimetric, was
designed by Chen and colleagues for the detection of palladium (II) ions
at 335 nm to 429 nm). At the same time the probe detected either way the presence of Cu ions but that can be used
2+ in living
cells.
couldThis oxime-ether
be used only forcoumarin probe (189,
the fluorescent sensingFigure
of Al61)3+exhibited
. A further a strong greenexploitation
interesting fluorescenceofwithprobean
emission peak at 500 nm. When palladium(II) was added to the solution
188 (Figure 60) was the fluorescent and colorimetric identification of Cys, Hcy and GSH when it waswith compound 189, the
fluorescence
complexed with intensity
copper ations.
500 nm decreased consequently, until 2 equivalents of Pd2+ were reached; at
that point
Despite thethe
fluorescence
key role of was almost
chemical completely
species quenched,
like copper which could they
and hypochlorite, clearly
arebenotobserved
the only with
ions
the naked eye. A linear fit between fluorescence and palladium (II) concentration
valuable for detection. For this reason, a dual coumarin probe, fluorescent and colorimetric, was observed wasin
the range 0.0-8.0
designed by Chen μM, while
and the detection
colleagues for thelimit was measured
detection to be (II)
of palladium 40 nM,
ionswhich
that canis far
be lower
used inthan the
living
threshold for palladium content in drugs (5.0 ppm to 10.0 ppm - 47.0 mM
cells. This oxime-ether coumarin probe (189, Figure 61) exhibited a strong green fluorescence with to 94.0 mM) specified by
the World Health Organization [245].
an emission peak at 500 nm. When palladium(II) was added to the solution with compound 189, the
It is alsointensity
fluorescence noteworthy at 500thenmdevelopment of thioacetalised
decreased consequently, untilcoumarin
2 equivalents based offluorescent probes for
Pd2+ were reached; at
the detection of mercury (II), a hazardous ion both for human health and reproduction.
that point the fluorescence was almost completely quenched, which could clearly be observed with the Cheng and
co-workers
naked eye. exploited
A linear fit the known fluorescence
between Hg2+ promoted anddeprotection
palladium (II)reaction of dithioacetals
concentration to design
was observed intwo
the
novel reactive fluorescent probes (190,191, Figure 61) that showed a different
range 0.0–8.0 µM, while the detection limit was measured to be 40 nM, which is far lower than the behavior due to the
different
thresholdchemical structures:
for palladium content 190 indisplayed
drugs (5.0remarkable
ppm to 10.0fluorescence
ppm - 47.0 mM quenching withspecified
to 94.0 mM) the addition of
by the
World Health Organization [245].
It is also noteworthy the development of thioacetalised coumarin based fluorescent probes for
the detection of mercury (II), a hazardous ion both for human health and reproduction. Cheng and
co-workers exploited the known Hg2+ promoted deprotection reaction of dithioacetals to design two
novel reactive fluorescent probes (190, 191, Figure 61) that showed a different behavior due to the
different chemical structures: 190 displayed remarkable fluorescence quenching with the addition
of Hg2+ ions while, in the presence of mercury ions, 191 displayed ratiometric fluorogenic and
chromogenic response [246].
It should not be forgotten an on-off fluorescent probe for the tracking of iron (III) ions based on
7-hydroxy-2-oxo-N-(pyridin-2-ylmethyl)chromene-3-carboxamide. In their work, Warrier and Kharkar
demonstrated that compound 192 (Figure 61) was selective towards Fe3+ ions and exhibited high
fluorescence emission profile at 447 nm. The presence of other ions did not interfere with the detection
of iron (III) ions and the limit of detection was found to be 0.76 µM. Moreover, cell imaging and MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay proved the potential utility of
probe 192 as cell-permeable chemosensor of Fe3+ in living cells [247].
The approach of Jiao and colleagues for the detection of fluoride ions was based on the linkage
between the coumarin scaffold and fluorescein in order to obtain a highly selective and sensitive
high fluorescence emission profile at 447 nm. The presence of other ions did not interfere with the
detection of iron (III) ions and the limit of detection was found to be 0.76 μM. Moreover, cell imaging
and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay proved the potential
utility of probe 192 as cell-permeable chemosensor of Fe3+ in living cells [247].
Int. J. Mol.
The Sci. 2020, 21, 4618
approach 40 of 81
of Jiao and colleagues for the detection of fluoride ions was based on the linkage
between the coumarin scaffold and fluorescein in order to obtain a highly selective and sensitive
fluorescent probe. The mechanism of F−- ions detection by compound 193 (Figure 61) was explained
fluorescent probe. The mechanism of F ions detection by compound 193 (Figure 61) was explained
and involved a desilylation reaction in the presence of fluoride ions. Moreover, a linear relationship
and involved a desilylation reaction in the presence of fluoride ions. Moreover, a linear relationship
between the ratio of emission intensities at 532 and 465 nm and−F− concentration over the range of 0–
between the ratio of emission intensities at 532 and 465 nm and F concentration over the range of 0–20
20 μΜ with a limit of detection of 0.025 μΜ was found [248]. Differently, Yao and co-workers
µM with a limit of detection of 0.025 µM was found [248]. Differently, Yao and co-workers exploited
exploited the capability of fluoride ions to form stable complex2+ with Ca2+ to design a novel fluorescent
the capability of fluoride ions to form stable complex with Ca to design a novel fluorescent sensor
sensor (194, Figure 61), synthesized from the combination of mandelic acid with 7-hydroxy-8-
(194, Figure 61), synthesized from the combination of mandelic acid with 7-hydroxy-8-formylcoumarin
formylcoumarin through a hydrazine hydrate bridge, in order to selectively identify these two ionic
through a hydrazine hydrate bridge, in order to selectively identify these two ionic species over other
species over other metal ions [249]. The fluorescence spectrum of compound 194 clearly increased
metal ions [249]. The fluorescence spectrum of compound 194 clearly increased when calcium ions
when calcium ions were added to the solution with a limit of detection of 5.81 × 10-7 M, while, once
were added to the solution with a limit of detection of 5.81 × 10−7 M, while, once the complex between
the complex between the probe and Ca2+ ions was obtained, the addition of fluoride ions to the
the probe and Ca2+ ions was obtained, the addition of fluoride ions to the solution lead to the turn-off of
solution lead to the turn-off of the fluorescence response−7with a limit of detection 4.28 × 10-7 M.
the fluorescence response with a limit of detection 4.28 × 10 M. Moreover, bio-imaging studies were
Moreover, bio-imaging studies were performed in order to assure the possibility to exploit this novel
performed in order to assure the possibility to exploit this novel chemosensor for the identification of
chemosensor for the identification of Ca2+ and F- ions in vivo, with positive outcome.
Ca2+ and F− ions in vivo, with positive outcome.
Finally, Reddy and Choi designed and synthesized three dicyanovinylcoumarin probes as turn-
Finally, Reddy and Choi designed and synthesized three dicyanovinylcoumarin probes as turn-on
on fluorescent sensor for the detection of CN - ions among other anions [250]. Within the different
fluorescent sensor for the detection of CN− ions among other anions [250]. Within the different
synthesized probes only compound 195 (Figure 61) showed a remarkable increase of the fluorescence
synthesized probes only compound 195 (Figure 61) showed a remarkable increase of the fluorescence
in presence of fluoride and cyanide ions with an interesting sensibility towards CN- ions: the limit of
in presence of fluoride and cyanide ions with an interesting sensibility towards CN− ions: the limit
detection was up to 11.4 nM, lower than the maximum level in drinkable water according to WHO
of detection was up to 11.4 nM, lower than the maximum level in drinkable water according to
guidelines.
WHO guidelines.
Chemical structure
Figure 61. Chemical
Figure structure of
of different
different coumarin fluorescent probes for the detection
detection of
of ions.
ions.
3.2. Food
3.2. Food Systems
Systems
Coumarins have
Coumarins havefound important
found applications
important also inalso
applications the agri-food sector. In fact,
in the agri-food the antimicrobial
sector. In fact, the
antimicrobial activity characterizing these natural compounds could be exploited or
activity characterizing these natural compounds could be exploited for food preservation forforfood
the
treatment of plant pathogens, infections in aquaculture or biofouling caused by eukaryotic organisms.
In addition, because coumarin scaffold has been used in fluorescent probing, natural coumarins might
be used in the detection of some substances in food samples. For instance, Zhang and collaborators
developed a new near-infrared probe constituted by a conjugated coumarin-indolium system, for
rapid, colorimetric and ratiometric fluorescent detection of bisulfite and sulfite anions [251]. (Bi)sulfite
anions (HSO3 − /SO3 2− ) are widely used as preservative for foods and beverages in order to prevent
oxidation, browning and microbial reaction during products’ life cycle [252,253]. Unfortunately, high
doses of (bi)sulfites can cause asthma or other allergic reactions. Some individuals are very sensitive
even to low levels of these anions [254]. In addition, sulfur dioxide (SO2 ) is one of the most distributed
pollutants and it has a relevant impact on human health [252], [253,255]. An efficient tool to detect
and sulfite anions [251]. (Bi)sulfite anions (HSO3−/SO32−) are widely used as preservative for foods and
beverages in order to prevent oxidation, browning and microbial reaction during products’ life cycle
[252,253]. Unfortunately, high doses of (bi)sulfites can cause asthma or other allergic reactions. Some
individuals are very sensitive even to low levels of these anions [254]. In addition, sulfur dioxide
(SO2) is one of the most distributed pollutants and it has a relevant impact on human health [252],
Int. J. Mol. Sci. 2020, 21, 4618 41 of 81
[253,255]. An efficient tool to detect such molecules is provided by fluorescent probes; to date,
different probes for HSO3−/SO32− have been designed but many of them are intensity-based, which
means
such that the signal
molecules outputby
is provided canfluorescent
be conditionedprobes;by to
different factors such
date, different probes for HSO3 − /SO
as instrumental 2− have
efficiency,
3
probedesigned
been concentration, but many environmental
of them areconditions. In addition,
intensity-based, whichmany means of that
thesethe
probes
signalshow output emissions
can be
only in the visible
conditioned region
by different and some
factors such asof instrumental
them need ultraviolet
efficiency,excitation, having limited
probe concentration, biological
environmental
applications.InThus,
conditions. new, more
addition, manyefficient
of theseprobes
probesare required.
show emissionsIn Zhang’s
only inwork, a conjugated
the visible region coumarin-
and some
indolium
of them need system with intramolecular
ultraviolet excitation, having charge
limitedtransfer (ICT)
biological effect was Thus,
applications. developed,
new, more merging an
efficient
electron-donating
probes are required. 7-diethylamino
In Zhang’s work, coumarin moiety and
a conjugated an electron-withdrawing
coumarin-indolium system with positively charged
intramolecular
indolium
charge derivative,
transfer (ICT) connected
effect was through
developed, an ethylene
merging linker (Figure 62). The7-diethylamino
an electron-donating whole system resulted coumarin in
a typical
moiety and ‘push-pull’ large conjugation
an electron-withdrawing dye system
positively charged with ICT properties.
indolium derivative,The so-designed
connected through probe
an
showed NIR
ethylene linker fluorescence
(Figure 62).(667 Thenm)wholeandsystem
a rapid, highlyinselective
resulted a typicaland sensitivelarge
‘push-pull’ detection processdye
conjugation for
HSO3−/SO
system 32- in
with ICTaqueous solution
properties. The under mild conditions;
so-designed probe showed in addition, this probe(667
NIR fluorescence exhibited
nm) and significant
a rapid,
3 /SO3 upon
colorimetric,
highly selective NIR andfluorescent and ratiometric
sensitive detection processsignal
for HSO −
responses 2− one excitation
in aqueous solutionwave underand milda
detection lower
conditions; in addition, ∼27 probe
limit of this nM. This probe can
exhibited be applied
significant to detect HSO
colorimetric, −/SO32- in real
NIR3fluorescent andfood samples,
ratiometric
serum responses
signal samples and upon living cells. When
one excitation wavetested
andona detection
real food lower
samples (sugar,
limit of ∼27soft
nM. sugar,
This crystal
probe can sugar
be
3 /SO3 this
and wine
applied toin aqueous
detect −
HSOsolution) 2− probe
in real foodproved
samples, to be ablesamples
serum to determine HSO3cells.
and living - withWhen
good recovery.
tested on
In addition,
real food samplesa paper test strip
(sugar, was developed,
soft sugar, crystal sugarsimply
and bywinewetting a stripsolution)
in aqueous of neutral filter
this probe paper
provedwithtoa
solution
be able toofdetermine
the probeHSO −
in methanol (200 μM). The result was a deep blue test paper
wasready for use:simply
when
3 with good recovery. In addition, a paper test strip developed,
a HSO
by 3− solution
wetting a stripisofspotted
neutralon the paper
filter test paper,
with rapid colorofchanges
a solution the probe caninbemethanol
observed,(200even µM).if many other
The result
ions aare
was present
deep blue in thepaper
test sample. Different
ready for use:colors a HSO3 − solution
whencorrespond to different concentrations
is spotted on the test of HSO
paper, 3−. rapid
Thus,
probechanges
color 196 (Figure can62)be can be usedeven
observed, to develop
if manya cheap, easy-to-prepare
other ions are present in and easy-to-use
the paper test
sample. Different strip
colors
system for the
correspond of (bi)sulfites.of HSO3 − . Thus, probe 196 (Figure 62) can be used to develop a
detectionconcentrations
to different
cheap, easy-to-prepare and easy-to-use paper test strip system for the detection of (bi)sulfites.
Figure 62. Chemical structure of probe 196.
A more recent example is aFigure

probe62. Chemical structure
developed of probe
by Nair and 196.
collaborators, able to selectively detect
−
the amphiphilic bisulfate ion (HSO4 ) in edible plant foods, dog urine and drugs [256]. Bisulfate
A more recent example is a probe developed by Nair and collaborators, able to selectively detect
consumption normally takes place through the ingestion of different edible plants, such as cabbage,
the amphiphilic bisulfate ion (HSO4−) in edible plant foods, dog urine and drugs [256]. Bisulfate
broccoli, Brussels sprouts, horseradish or seeds (black and white mustard, for instance). These plants
consumption normally takes place through the ingestion of different edible plants, such as cabbage,
contain glucosinolates [257,258] that are hydrolyzed in our organism by the enzyme myrosinase,
broccoli, Brussels sprouts, horseradish or seeds (black and white mustard, for instance). These plants
thus producing bisulfate ion [259]. In addition, bisulfate salts of many APIs are currently on market,
contain glucosinolates [257,258] that are hydrolyzed in our organism by the enzyme myrosinase, thus
constituting another source of bisulfate ions [260]. When trying to evaluate the actual concentration of
producing bisulfate ion [259]. In addition, bisulfate salts of many APIs are currently on market,
HSO4 − , it is important to take account of the deprotonation equilibrium between HSO4 − and SO4 2− ,
constituting another source of bisulfate ions [260]. When trying to evaluate the actual concentration
using a highly selective probe able to discriminate between these two ions. Nair and co-workers
of HSO4−, it is important to take account of the deprotonation equilibrium between HSO4− and SO42-,
developed two fully water-soluble probes, coumarin-integrated glycine (CG) and coumarin-integrated
alanine (CA) zwitterions, for the selective detection of HSO4 − at picomolar level (from 50 to 325 pM)
(Figure 63).
using a highly selective probe able to discriminate between these two ions. Nair and co-workers
developed two fully water-soluble probes, coumarin-integrated glycine (CG) and coumarin-
integrated
Int. J. Mol. Sci.alanine
2020, 21, (CA)
4618 zwitterions, for the selective detection of HSO4 at picomolar level (from
−
42 of 50
81
to 325 pM) (Figure 63).
Figure 63. Chemical structure of CG and

Figure 63. and CA
CA probes.
probes.
Glycine
Glycine and and alanine
alanine can can interact
interact selectively
selectively with with thethe target
target through
through H-bond,
H-bond, due due to to their
their
zwitterionic nature at physiological pH, whereas the 7-hydroxycoumarin
zwitterionic nature at physiological pH, whereas the 7-hydroxycoumarin moiety constitutes a good moiety constitutes a good
fluorophore
fluorophore probe,
probe, thanks
thanks to to its
its biocompatibility,
biocompatibility, non-toxicity
non-toxicity and and water
water solubility.
solubility. The The CG/CA
CG/CA
probes
probes proved
provedto tobe beable
abletotopenetrate
penetrateand andstain
stainliving
living cell.
cell.When
When different
differentunknown
unknown concentrations
concentrations of
clopidogrel bisulfate were added to a water solution of CG/CA, it
of clopidogrel bisulfate were added to a water solution of CG/CA, it was possible to precisely was possible to precisely measure such
concentration
measure suchby measuring the
concentration by emission
measuring intensity
the emissionof eachintensity
sample. of Confirmation
each sample.ofConfirmation
the experimentallyof the
observed values observed
experimentally with the theoretically
values with calculated ones supported
the theoretically calculated the accuracy
ones supportedof thethe presented
accuracy method.
of the
CG/CA
presentedprobes wereCG/CA
method. also tested probeson foodweresamples:
also tested wateron extracts
food samples:of cruciferous plant foods
water extracts (cabbage,
of cruciferous
broccoli,
plant foodsmustard
(cabbage, seeds, carrots)
broccoli, were added
mustard seeds, to aqueous
carrots) were solutions
added toofaqueous
the probes. Again,
solutions of titration
the probes. of
bisulfate ions was performed by emission measurements—addition
Again, titration of bisulfate ions was performed by emission measurements—addition of increasing of increasing volumes of aqueous
food sapsof
volumes caused
aqueous a growing
food saps reduction
causedofaprobe’s
growing emission.
reduction Cucumber
of probe’s andemission.
fenugreek were usedand
Cucumber as
controls,
fenugreekasweretheyuseddo not as contain
controls,bisulfates.
as they doEventually,
not containbisulfate
bisulfates. content was measured
Eventually, bisulfate in pet urine
content was
samples (bisulfate is one of the common components of pet foods):
measured in pet urine samples (bisulfate is one of the common components of pet foods): urine urine samples were collected from
two adultwere
samples dogscollected
and were treated
from two with
adultaqueous
dogs andsolution of CG/CA;
were treated with aaqueous
reduction of probe
solution emissiona
of CG/CA;
was noticed,ofrevealing the presence −
reduction probe emission wasofnoticed,
HSO4 (quenching
revealing the was presence
proportional of toHSObisulfate quantity).was
4− (quenching In
conclusion,
proportional to bisulfate quantity). In conclusion, CG and CA probes demonstrated the ability at
CG and CA probes demonstrated the ability to detect bisulfate ions in aqueous solution to
pH = 7.4,
detect wherein
bisulfate HSO
ions
− concentration was 105.4 lower than that of SO
in4 aqueous solution at pH = 7.4, wherein HSO4− 4concentration
2− , at concentration as
was 105.4low loweras
50 pM,
than even
that in presence
of SO of other ions.
42-, at concentration as low as 50 pM, even in presence of other ions.
With
With regards to the antimicrobial activity
regards to the antimicrobial activity of of coumarin
coumarin scaffold,
scaffold, some
some studies
studies havehave been
been recently
recently
carried
carried out,
out, showing
showing the the potential
potential of of natural
natural coumarins
coumarins as as food
food preservatives.
preservatives. Yang Yang andand co-workers
co-workers
have
have studied
studied the the antimicrobial
antimicrobial activity activity of of eighteen
eighteen naturalnatural compounds
compounds againstagainst R. Solanacearum [261],
R. Solanacearum [261],
aa bacterium responsible for the wilting of different plants such as tobacco,
bacterium responsible for the wilting of different plants such as tobacco, tomato, potato in tomato, potato in (sub)tropical
regions, causing
(sub)tropical significant
regions, causing economic
significant losses [262,263].
economic losses [262,263].
Among
Among them, four coumarins (Figure 64) showed an
them, four coumarins (Figure 64) showed an antibacterial
antibacterial activity
activity stronger
stronger thanthan that
that of
of
thiodiazole
thiodiazole copper
copper treatment
treatment (antibacterial
(antibacterial rate rate (MBC/MIC)
(MBC/MIC) of of 63.3%).
63.3%). Daphnetin
Daphnetin showed showed the the highest
highest
activity,
activity,followed
followed by byxanthotol
xanthotoland esculetin (antibacterial
and esculetin rate 97.43%,
(antibacterial rate80.12%
97.43%, and80.12%
71.44%, respectively).
and 71.44%,
Antibacterial activity seemed to be enhanced by
respectively). Antibacterial activity seemed to be enhanced by C6, C7 or C8 C6, C7 or C8 substitution, so hydroxycoumarins
substitution, so
umbelliferone,
hydroxycoumarins esculetin and daphnetin
umbelliferone, were
esculetin andselected
daphnetin for further investigation
were selected of theinvestigation
for further mechanism of of
action. Hydroxycoumarins were tested from 10 to 100 mg/L concentrations
the mechanism of action. Hydroxycoumarins were tested from 10 to 100 mg/L concentrations and from and from results it was
clear that
results it the
wasgoodclearactivity
that the of umbelliferone
good activity can be enhanced bycan
of umbelliferone thebeadditional
enhanced hydroxylation
by the additionalof C6
position (esculetin),
hydroxylation of C6 whereas
position even better results
(esculetin), can be even
whereas achieved betterby the dihydroxylation
results can be achieved of C7 andby theC8
positions (daphnetin). TEM images of R. solanacearum showed
dihydroxylation of C7 and C8 positions (daphnetin). TEM images of R. solanacearum showed that that daphnetin and esculetin caused
irreversible
daphnetin and damages
esculetin to the cell membrane,
caused irreversible whereas
damagesumbelliferone must follow
to the cell membrane, whereasa different path in
umbelliferone
inducing cell damage. It is worth noting that hydroxycoumarins showed
must follow a different path in inducing cell damage. It is worth noting that hydroxycoumarins showed very low cytotoxicity on
human cells and have no effects on tobacco seeds’ germination. Furthermore,
very low cytotoxicity on human cells and have no effects on tobacco seeds’ germination. Furthermore, because R. solanacearum
forms
becausebiofilm-like
R. solanacearum aggregations on host’s aggregations
forms biofilm-like roots, facilitating bacterial
on host’s roots,infection
facilitating [264], Yanginfection
bacterial and his
group speculated that hydroxycoumarins may interfere with biofilm
[264], Yang and his group speculated that hydroxycoumarins may interfere with biofilm formation. In formation. In fact, daphnetin,
umbelliferone
fact, daphnetin,and esculetin (100
umbelliferone andmg/L) reduced
esculetin (100 mg/L)biofilm formation
reduced by 99.22%,
biofilm formation 85.20% and 93.90%,
by 99.22%, 85.20%
respectively,
and 93.90%, probably
respectively, influencing
probably theinfluencing
swimming motilitythe swimming of the bacterium.
motility ofThus, the expression
the bacterium. Thus, of the
the
regulating and structural flagellar genes fliA, flhC and flhD in presence of daphnetin, umbelliferone
and esculetin was evaluated and it turned out that the expression of fliA and flhC was significantly
repressed by the mentioned compounds.
expression of the regulating and structural flagellar genes fliA, flhC and flhD in presence of daphnetin,
expression of the regulating and structural flagellar genes fliA, flhC and flhD in presence of daphnetin,
umbelliferone and esculetin was evaluated and it turned out that the expression of fliA and flhC was
umbelliferone
Int.
and 21,
J. Mol. Sci. 2020,
esculetin was evaluated and it turned out that the expression of fliA and flhC was
4618 by the mentioned compounds. 43 of 81
significantly repressed
significantly repressed by the mentioned compounds.
Figure 64. Chemical

Figure structure
64. Chemical of coumarins
structure umbelliferone,
of coumarins esculetin,
umbelliferone, daphnetin
esculetin, and and
daphnetin xanthotol.
xanthotol.
Figure 64. Chemical structure of coumarins umbelliferone, esculetin, daphnetin and xanthotol.
Another
Anotherinteresting
interestingbiological
biologicalactivity
activityascribed
ascribedto tothe
thecoumarin
coumarinnucleus
nucleusisisits itsantifungal
antifungalactivity,
activity,
Another
which interesting biological activity ascribed to the coumarin nucleus is its antifungal activity,
whichcan canbebeuseful
usefulnot
notonly
onlyinin
thethemedicinal
medicinal field butbut
field also in the
also in theagricultural
agricultural one. As As
one. far far
as the latter
as the is
latter
which can be there
concerned, usefulhas
notbeen
onlyaninincreasing
the medicinal fieldinbut
interest thealso in the agricultural
exploitation of coumarin one.scaffold
As far asfor the
the latter
design
is concerned, there has been an increasing interest in the exploitation of coumarin scaffold for the design
is concerned,
and there has been an increasing interest in the exploitation of coumarin scaffold for the design
andsynthesis
synthesisofofnovelnovelfungicides
fungicidesuseful
usefulininthe thetreatment
treatmentofofmany manyplantplantdiseases.
diseases.Many
Manypathogenic
pathogenic
and synthesis
fungi of novel fungicides useful
crops,inlimiting
the treatment of many plant diseases. Many pathogenic
fungistill
stillcause
causemassive
massive death
death ofof crops, limiting production
production andcausing
and causing significant
significant financial
financial losses.
losses. To
fungi
To still
date, cause
farmersmassive
deal death
with of
thiscrops,
problem limiting
by production
using chemical and causing
fungicides significant
but such financial
solution losses. To
comes with
date, farmers deal with this problem by using chemical fungicides but such solution comes with some
date,
somefarmers deal with
drawbacks: thethis
longproblem by using chemicalthesefungicides
chemicalsbut such solution comes with some
drawbacks: the long andand extensive
extensive useuse ofofthese chemicals may
may cause
cause environmental
environmental pollution,
pollution,
drawbacks:
accumulation the long and extensive use ofplants
these chemicals may causefungi. environmental pollution,
accumulationofofpesticides
pesticidesresidues
residuesininthe the plantsand anddrug
drugresistance
resistanceby by fungi.Thus,
Thus,there
thereisisananurgent
urgent
accumulation
need for of pesticides
alternative (and residues
green) in the
solutions.plants
In and
2019, drug resistance
Ramìrez-Pelayo by fungi.
and his Thus, there
collaborators is an urgentthe
studied
need for alternative (and green) solutions. In 2019, Ramìrez-Pelayo and his collaborators studied the
need for alternative (and green)contained
coumarin-based solutions. Inthe 2019, Ramìrez-Pelayo and his collaborators studied the
coumarin-basedcompounds compounds containedin in thepeelpeelofofcitrus
citrusgrown
grownininColombia
Colombia[265]. [265].Six
Sixcoumarins
coumarins
coumarin-based
were compounds contained in the antifungal
peel of citrus grown in Colombia [265]. Six coumarins
wereisolated
isolated(197–202,
(197–202,Figure
Figure65)65)and
andtheir
their antifungalactivity,
activity,ininterms
termsofofmycelial
mycelialgrowth
growthand andspore
spore
were isolated
germination (197–202, Figure 65) and their antifungal activity, in terms of mycelial growth andsp.). spore
germination inhibition, was evaluated against the fungus causing anthracnose (Colletotrichum sp.).The
inhibition, was evaluated against the fungus causing anthracnose (Colletotrichum The
germination
production inhibition, was evaluated against the fungus causing anthracnose (Colletotrichum sp.). The
productionofoforanges,
oranges,mandarins,
mandarins,grapefruit,
grapefruit,lemons,
lemons,limeslimesisislimited
limitedby bythetheaction
actionofofmicroorganisms
microorganisms
production
able of oranges, mandarins, grapefruit, lemons, condition
limes is limited as by the action of microorganisms
abletotoproliferate
proliferateininsuch
suchfruits
fruitsbecause
becauseofofpeculiar
peculiar conditionsuch such ashighhighnutrient
nutrientcontent,
content,humidity
humidity
able
andto proliferate
low pH in
values.such fruits because
Nevertheless, of
these peculiar
plants condition
can defend such as high
themselves nutrient
from content, humidity
pathogens thanks
and low pH values. Nevertheless, these plants can defend themselves from pathogens thanks to to
the
andthelow pH
production values. Nevertheless, these plants can defend themselves from pathogens thanks to the
production of of antimicrobialcompounds
antimicrobial compoundswhich whichcan can bebe constitutive
constitutive (phytoanticipins)
(phytoanticipins) or orinduced
induced
production
(phytoalexins) of antimicrobial compounds which can be constitutive (phytoanticipins) or induced
(phytoalexins)[266,267].
[266,267].
(phytoalexins) [266,267].
Figure 65. Structures of compounds isolated from Citrus peel.

As already mentioned, from the methanol extracts of C. latifolia and C. aurantifolia peels six
compounds were isolated—5-geranyloxy-7-methoxycoumarin (197), bergamottin (198), bergapten
As already mentioned, from the methanol extracts of C. latifolia and C. aurantifolia peels six
compounds
Int. were
J. Mol. Sci. 2020, 21, isolated—5-geranyloxy-7-methoxycoumarin
4618 (197), bergamottin (198), bergapten 44 of 81
(199), isopimpinellin (200), limettin (201) and oxypeucedanin hydrate (202) and their activity was
compared to that of umbelliferone, scoparone and scopoletin. Compounds 197–201 were tested at
(199),
0.25, 0.50isopimpinellin
and 1.00 mM, (200), limettin
whereas 202(201) and oxypeucedanin
was tested hydrate (202)
at 1.00 mM concentration. and their activity
Carbendazin and thymol was
compared to that of umbelliferone, scoparone and scopoletin.
were used as reference compounds (0.5 mM). The results showed that compounds 197–202 Compounds 197–201 were tested at 0.25,
0.50 and 1.00 inhibited
significantly mM, whereas 202 was
mycelial growthtestedof at 1.00 mM concentration.
Colletotrichum sp., the activity Carbendazin and thymolto
being proportional were
the
used as reference compounds (0.5 mM). The results showed
dose used. The highest inhibition was exhibited by compounds 199 and 201 (32% and 25%, that compounds 197–202 significantly
inhibited
respectively); mycelial growth
therefore, Colletotrichum
theoffungistatic effectsp.,
of the activity
different being proportional
mixtures of 199 and 201 to (0.25
the dose
mMused. The
(192):0.75
highest
mM (201), inhibition
0.50 mM was exhibitedmM
(199):0.50 by compounds
(201) and 0.75 199mMand(199):0.25
201 (32% mM and 25%,(201))respectively);
was investigated, therefore,
eachthe of
fungistatic effect of different mixtures of 199 and 201 (0.25 mM (192):0.75
them showing enhanced activity respect to both the individual compounds. Furthermore, the activity mM (201), 0.50 mM (199):0.50
mM
of 199,(201)201andand0.75 mMmixtures
their (199):0.25was mMcompared
(201)) wasto investigated,
that of the each of them scoparone,
phytolaxins showing enhanced scopoletin activity
and
respect to both the individual compounds. Furthermore, the activity
umbelliferone; the results showed that phytolaxins were just slightly more active than the isolated of 199, 201 and their mixtures
was compared
compounds, to that of
although thethe phytolaxins
highest scoparone,
fungistatic activityscopoletin
was shown and byumbelliferone;
the mixture ofthe 201results
(0.75 mM) showedand
that phytolaxins
199 (0.25 mM). This were just slightly
mixture, along withmorecompounds
active than197, the 199
isolated
and 201,compounds,
was selected although
for thethe highest
evaluation
fungistatic
of the inhibitory activity effect
was shown on spore by the mixture of in
germination (0.75 mM) and
201comparison with199 (0.25 mM).scoparone
scopoletin, This mixture, and
along with compounds 197, 199 and 201, was selected
umbelliferone. Compounds 201 and 199 exhibited very good activity, with 96.7% and for the evaluation of the inhibitory effect
95.3%on
spore germination
inhibition, in comparison
respectively but thesewith scopoletin,
percentages scoparone
rapidly and umbelliferone.
decrease, being less than Compounds
5% after 24201 and
hours.
199 exhibited very good activity, with 96.7% and 95.3% inhibition, respectively
Again, the 199/201 mixture showed the highest activity causing a complete, long-lasting inhibition of but these percentages
rapidly decrease, being
spore germination, thusless than 5% after
suggesting 24 h. may
that there Again, bethe additivemixture
an 199/201 showedeffect
or synergistic the highest
between activity
these
causing a complete, long-lasting inhibition of spore germination,
compounds. Therefore, coumarins and furanocoumarins may be useful scaffolds in the design of newthus suggesting that there may be an
additive or synergistic effect between these compounds. Therefore,
antifungal agents. In 2018, Yu and co-workers designed a series of coumarin-3-carboxamide coumarins and furanocoumarins
may be useful
derivatives andscaffolds
evaluated in the design
their of newactivity
antifungal antifungal agents.
against In 2018,
Botrytis Yu and
cinerea, co-workers
Alternaria solani,designed
Gibberella a
series of coumarin-3-carboxamide derivatives and evaluated their antifungal
zeae, Rhizoctonia solani, Cucumber anthrax and Alternaria leaf spot [268]. These phytopathogenic fungi activity against Botrytis
cinerea, Alternariainsolani,
are all relevant Gibberella
agriculture, zeae, Rhizoctonia
because they can causesolani,significant
Cucumber anthrax
losses in and Alternaria
crops leaf spot [268].
and investments. In
These phytopathogenic fungi are all relevant in agriculture, because
this work, two different scaffolds were merged in order to design a new category of more effective they can cause significant losses in
crops and investments.
fungicides. In fact, the use Inofthis work, two
carboxylic aciddifferent scaffolds
amides (CAA) werecontext
in this merged in order to designbecause
is well-established, a new
category of more effective
amide fungicides have been fungicides.
used for In fact,50the
over use [269].
years of carboxylic acid amides
More recently, new(CAA)
amide in this context
fungicides with is
well-established, because amide fungicides have been used for over
high activity and broad spectrum of action were developed (e.g., Fluopyram, Bixafen, Sedaxane, 50 years [269]. More recently, new
amide
Isopyrazam,fungicides with highand
Penthiopirad activity and broad
Boscalid) [1,270].spectrum
In addition,of action
hydrazidewere scaffolds
developed (e.g.,
have beenFluopyram,
found to
Bixafen, Sedaxane,
have interesting Isopyrazam,
biological Penthiopirad and
and pharmacological Boscalid)
activities [1,270]. Similarly,
[271,272]. In addition, thehydrazide
antifungalscaffolds
activity
have been found to have interesting biological and pharmacological
of coumarin nucleus is well known (paragraph 0). Considering these elements, the authors decided activities [271,272]. Similarly, the
antifungal
to combineactivitythe coumarinof coumarin
and thenucleus is well known (paragraph
carboxamide/hydrazide scaffolds with 0). Considering these elements,
the aim of synthesizing high-
the authors decided to combine the coumarin and the carboxamide/hydrazide
performance fungicides. All the synthesized compounds were tested and their antifungal activities scaffolds with the aim
of synthesizing high-performance fungicides. All the synthesized
against the mentioned phytopathogenic fungi were evaluated. At 100 μg/mL concentration, most of compounds were tested and their
antifungal activities against
the tested compounds showed the poor
mentioned
antifungalphytopathogenic
activity. Onlyfungi compoundswere evaluated.
203 and 204 At (Figure
100 µg/mL 66)
concentration, most of the tested compounds showed poor antifungal
showed EC50 values of 1.57 μg/mL and 1.65 μg/mL, respectively, against Botrytis cinerea, proving to activity. Only compounds 203
and 204 (Figure
be equivalent to66)
theshowed
reference ECBoscalid
50 values(0.51of 1.57 µg/mL
μg/mL), and 1.65
whereas 203 (EC50 respectively,
µg/mL, = 1.80 μg/mL)against resulted Botrytis
more
cinerea,
active than proving to be(EC
Boscalid equivalent to the reference
50 = 2.98 μg/mL) solani.µg/mL), whereas 203 (EC50 = 1.80
Boscalid (0.51
against Rhizoctonia
µg/mL) resulted more active than Boscalid (EC50 = 2.98 µg/mL) against Rhizoctonia solani.
Figure 66. Chemical structure of compounds 203 and 204.

Figure 66. Chemical structure of compounds 203 and 204.
Starting from these data it was possible to suggest some structure-activity relationships: the
replacement
Starting of amide
from bond
these datawith a hydrazide
it was possible to bond led to
suggest somean improvement in antifungal
structure-activity activity
relationships: the
spectrum. Furthermore, coumarins bearing amide group resulted to be remarkably
replacement of amide bond with a hydrazide bond led to an improvement in antifungal activity more active
against
spectrum.Botrytis cinerea andcoumarins
Furthermore, Rhizoctonia bearing
solani, whereas
amide against Alternariato
group resulted solani, Gibberella zeae,
be remarkably moreCucumber
active
anthrax and Alternaria leaf spot they showed less potency. It is noteworthy that compounds
against Botrytis cinerea and Rhizoctonia solani, whereas against Alternaria solani, Gibberella zeae, bearing an
electron-withdrawing group did not show any activity, whereas compounds with an electro-donating
group were active against Alternaria leaf spot. The addition to the coumarin nucleus of Cl/F- substituted
Cucumber anthrax and Alternaria leaf spot they showed less potency. It is noteworthy that compounds
bearing an electron-withdrawing group did not show any activity, whereas compounds with an
electro-donating group were active against Alternaria leaf spot. The addition to the coumarin nucleus
of Cl/F- substituted phenylidrazine gave compounds active on Cucumber anthrax. A similar approach
Int. J. Mol. Sci. 2020, 21, 4618 45 of 81
is the one followed by Yang and collaborators, who decided to exploit coumarin nucleus for the
functionalization of chitosan, in order to create more effective chitosan-based fungicides [273]. In fact,
chitosan is becoming
phenylidrazine more and more
gave compounds activeappreciated
on Cucumberdue to itsA antimicrobial
anthrax. similar approach activity, lowfollowed
is the one toxicity,by
biodegradability, biocompatibility
Yang and collaborators, and to
who decided film forming
exploit activity.
coumarin However,
nucleus its functionalization
for the application as fungicide is
of chitosan,
limited by its low solubility and weaker activity respect to the on-market fungicides.
in order to create more effective chitosan-based fungicides [273]. In fact, chitosan is becoming more and In this work,
four
morecoumarin-functionalized chitosan derivatives
appreciated due to its antimicrobial activity, (205a–d, Figure
low toxicity, 67), were synthesized
biodegradability, and tested
biocompatibility and
against the phytopathogens
film forming activity. However, Alternaria solani sorauer,
its application Fusarium
as fungicide oxysporum
is limited by its lowf.sp. vasinfectum
solubility and
and weaker
Fusarium
activitymoniliforme
respect to by theevaluating
on-marketthe mycelial growth
fungicides. In thisrate in vitro.
work, four At 1.0 mg/mL, compoundschitosan
coumarin-functionalized 205a–
d derivatives
showed inhibitory(205a–d,activity against
Figure 67), wereA.synthesized
solani, following the sequence
and tested against the 205d > 205b > 205cAlternaria
phytopathogens > 205a
(38.25%, 51.23%, 44.01% and 52, 78%, respectively).
solani sorauer, Fusarium oxysporum f.sp. vasinfectum and Fusarium moniliforme by evaluating the mycelial
growth rate in vitro. At 1.0 mg/mL, compounds 205a–d showed inhibitory activity against A. solani,
following the sequence 205d > 205b > 205c > 205a (38.25%, 51.23%, 44.01% and 52, 78%, respectively).
Figure 67.67.
Figure Chemical structure
Chemical of of
structure coumarin-functionalized chitosan
coumarin-functionalized 205a–d.
chitosan 205a–d.
Theseresults
These results suggested
suggested that
thatthe introduction
the introductionof halogens caused
of halogens an increase
caused in activity,
an increase in depending
activity,
on the number and the type of halogens. For instance, the 3,5-dichloro derivatives
depending on the number and the type of halogens. For instance, the 3,5-dichloro derivatives were were more active
against
more A. against
active solani than the correspondent
A. solani 5-Cl or 5-Br
than the correspondent derivatives,
5-Cl probably probably
or 5-Br derivatives, because ofbecause
an increase
of anof
hydrophobicity.
increase The four coumarin-chitosan
of hydrophobicity. derivatives derivatives
The four coumarin-chitosan showed higher activity
showed thanactivity
higher chitosanthan
alone
chitosan aloneF.also
also against oxysporum
against and F. moniliforme.
F. oxysporum and F.Compound
moniliforme.205d
Compound
showed an205d showed index
inhibitory an inhibitory
of 57.09%
against
index A. solani,
of 57.09% 77.24%
against A. against F. oxysporum
solani, 77.24% againstand 66.12% against
F. oxysporum F. moniliforme.
and 66.12% against F. moniliforme.
3.3.
3.3. Coumarin-Metal
Coumarin-Metal Complexes
Complexes
TheThegreat
great biological
biological potential
potentialofofcoumarins
coumarins might be even
might increased
be even by considering
increased the possibility
by considering the
to complex coumarin scaffold with other substances as, for example,
possibility to complex coumarin scaffold with other substances as, for example, some metals. In fact,some metals. In fact, it has
it has been proved that it is possible to enhance the activity of a certain drug simply binding it to a a
been proved that it is possible to enhance the activity of a certain drug simply binding it to
metallic-element
metallic-element [274];
[274]; moreover,
moreover, byby combining
combining known
known active
active moieties
moieties with
with metals,
metals, it it would
would bebe
possible to improve the parent compound with a certain selectivity
possible to improve the parent compound with a certain selectivity or even with a new mechanism or even with a new mechanism
ofof action.
action. Therefore,
Therefore, manymany groups
groups have
have already
already started
started to to explore
explore thisthis strategy,
strategy, aiming
aiming toto produce
produce
more active compounds, exploiting metals as copper, platinum, zinc
more active compounds, exploiting metals as copper, platinum, zinc or silver. In some cases, due to or silver. In some cases, due
its intrinsic potential, coumarin scaffold was exploited in this approach. Some examples are discussedare
to its intrinsic potential, coumarin scaffold was exploited in this approach. Some examples
discussed below.
below.
TheThe nature
nature ofof coumarin-copper
coumarin-copper complexes
complexes had
had already
already been
been evaluated
evaluated inin 2001
2001 byby Karaliota
Karaliota andand
co-workers, who synthesized and characterized a binuclear coumarin-3-carboxilate-copper
co-workers, who synthesized and characterized a binuclear coumarin-3-carboxilate-copper (II) (II) complex
(206) [275].
complex (206)In this In
[275]. work,
this thanks to multiple
work, thanks instrumental
to multiple characterization
instrumental by means
characterization of IR, of
by means Raman
IR,
Raman and NMR spectroscopy, the authors were able to identify the structure of the complex (Figure a
and NMR spectroscopy, the authors were able to identify the structure of the complex (Figure 68):
binuclear
68): molecule
a binuclear molecule[Cu[Cu2 (Cca) 4 (H42(H
2(Cca) O)22O)
], 2where copper
], where copperis coordinated
is coordinated bybyfour carboxylic
four carboxylicoxygens,
oxygens, one
from each Cca molecule and two water
one from each Cca molecule and two water molecules. molecules.
Int. J. Mol. Sci. 2020, 21, 4618 46 of 81
Figure 68. Chemical structure of coumarin-3-carboxilate-copper (II) complex (206).
A more recent Figure

Figure
work 68.by
68. Chemical
Chemical
MacLean structure
structure of coumarin-3-carboxilate-copper
et al. reports a series of copper (II) complexes (II) complexwith(206).
seven imine-
derived ligands (207–213, Figure 69) which were tested on MCF-7 human breast cancer cells to
A more
morerecentrecentwork workbybyMacLean
MacLean et al. reports a series of copper (II) complexes with imine-
seven
evaluate A their cytotoxicity [276]. In fact, the et al. reports
increase a series
of of copper
ROS levels might(II)promote
complexes with seven
oxidative stress-
imine-derived
derived ligands ligands
(207–213,(207–213, Figure 69) which were tested on MCF-7 human breast cancer cells
induced cancer cell death, thusFigure 69) which
representing were tested
an alternative on MCF-7
strategy for thehuman
treatment breast cancer
of some cells
forms to
to evaluate
evaluate theirtheir cytotoxicity
cytotoxicity [276].
[276]. In In the
fact, fact,increase
the increase
of ROS of levels
ROS levels
might might
promote promote
oxidative oxidative
stress-
of tumors [277–279]. Interestingly, it was found that the activity of such compounds depended also
stress-induced
induced cancercancer
cell cell death,
death, thus thus representing
representing an alternative strategythefor the treatment offorms
some
on their speciation; for example, 207, monomericanatalternative
neutral pH, strategy
showedforvaluable treatment of whereas
activity, some
forms of tumors [277–279]. Interestingly, it was found that the activity of such compounds depended
212 of
- a tumors
dinuclear [277–279]. Interestingly,
specie- resulted inactive. it was found that
Moreover, theirthe activity ofwas
cytotoxicity such notcompounds depended
related to their abilityalso
also on
on their their speciation;
speciation; for example,
for example, 207, monomeric
207, monomeric at neutral pH, showed valuable activity, whereas
to induce ROS generation. Such activity was showedatonly neutral pH, showed
by complex 207,valuable
whereasactivity,
210 andwhereas212
212
212 -
- a
a dinuclear
dinuclear specie-
specie- resulted
resulted inactive.
inactive. Moreover,
Moreover, their
their cytotoxicity
cytotoxicity was
was notnotrelated
relatedto their
to ability
their to
ability
exhibited limited ROS induction. 211 and 212 proved to be able to bind DNA but none of the tested
induce ROS
to induceshowed generation.
ROS generation. Such activity was showed only by complex 207, whereas 210 and 212 exhibited
compounds significantSuch activityactivity.
nuclease was showed only bythat
This means complex 207, whereas
imine-copper(II) 210 and 212
complexes
limited
exhibited ROS induction.
limited towards 211
ROS induction.and 212 proved
211 and to be
212act able
proved to bind DNA
to beofable but none of the tested compounds
showing cytotoxicity MCF-7 cells do not by means ROStogeneration
bind DNAorbut none of
nuclease the tested
activity,
showed
compounds significant
showed nuclease activity.
significant This
nuclease means that
activity. imine-copper(II)
This means complexes
that showing
imine-copper(II) cytotoxicity
complexes
being these two the mechanisms of action previously ascribed to copper (II) complexes [280]. In fact,
towards MCF-7
showing cytotoxicitycells do not act by means cells ofdoROS generation orofnuclease activity, or being theseactivity,
two the
complexes 208–210 actedtowards MCF-7
as superoxide dismutase not act
(SOD) byandmeans
catalaseROS generation
mimics. nuclease
This is noteworthy, if
mechanisms of action previously ascribed to copper (II) complexes [280]. In fact, complexes 208–210
we consider that in some cancer cells hypoxia induces an adaptive mechanism providing protectionfact,
being these two the mechanisms of action previously ascribed to copper (II) complexes [280]. In
acted as superoxide
complexes 208–210 dismutase
acted (SOD) and
as superoxide catalase (SOD)
dismutase mimics.and This is noteworthy,
catalase mimics. This if weisconsider
noteworthy, that in
against damage and oxidative stress. In this context, copper (II)-imine complexes acting as SOD if
some
we cancer that
consider cells inhypoxia induces an adaptive mechanism providing protection against damage and
mimics may constitute asome
validcancer
tool for cells
thehypoxia
treatment induces an adaptive
of hypoxic tumors.mechanism providing protection
oxidative stress. In this context, copper (II)-imine
against damage and oxidative stress. In this context, copper (II)-imine complexes acting as SOD mimics may
complexes actingconstitute
as SODa
valid tool for the treatment of hypoxic tumors.
mimics may constitute a valid tool for the treatment of hypoxic tumors.
Figure 69. Chemical structure of complexes 207–213.
In the same year, Figure

Qin and69. collaborators investigated
Chemical structure the207–213.
of complexes potential of platinum (II) complexes
against cisplatin resistant human lung adenocarcinoma (A549/DDP) and HeLa cells [281]. Cisplatin
In the
was same
used year, QinInand
as reference. Figure
this work,69. eleven
Chemical
collaborators structure of
investigated
complexes complexes
the 207–213.
potential
between platinumof platinum (II) complexes
(II) and quinoline-coumarin
against cisplatin(214–224,
derivatives resistantFigure
human 70)lung
wereadenocarcinoma (A549/DDP)
designed, synthesized and HeLa
and tested. All thecells
new[281]. Cisplatin
complexes showed
wasanusedIn
asthe same
reference. year,
In Qin
this and
work, collaborators
eleven complexesinvestigated
between the potential
platinum (II) of
and platinum (II) complexes
quinoline-coumarin
improved antineoplastic activity on A549/DDP and HeLa cells when compared to cis-Pt(DMSO)2 Cl2
against
and the cisplatin
derivatives (214–224,
parent resistant
Figure human
70) were
quinoline-coumarin lung adenocarcinoma
designed,
derivatives, synthesized
with IC50 (A549/DDP)
and tested.
values andAll
ranging HeLa
the cells
between new
100[281]. Cisplatin
complexes
nm and 10.33
was
showed used
µM (75.02 as± reference.
an improved1.18 µM In A549/DDP
for this work,
antineoplastic eleven
activity complexes
on
or 12.09 A549/DDP
± 0.24 µM between
and
for platinum
HeLa
HeLa). cells
In (II)
whenandcompared
addition, quinoline-coumarin
compounds to214–224
cis-
derivatives (214–224, Figure 70) were designed, synthesized and tested. All the new complexes
showed an improved antineoplastic activity on A549/DDP and HeLa cells when compared to cis-
100nm and 10.33 μM (75.02 ± 1.18 μM for A549/DDP or 12.09 ± 0.24 μM for HeLa). In addition,
compounds 214–224 displayed selectivity towards the mentioned cancer cells over other tumoral cell
lines and HL-7002 non-tumoral cell line.
Pt(DMSO)2Cl2 and the parent quinoline-coumarin derivatives, with IC50 values ranging between
Int. J. Mol.and
100nm Sci. 2020,
10.3321,μM
4618(75.02 ± 1.18 μM for A549/DDP or 12.09 ± 0.24 μM for HeLa). In addition,
47 of 81
compounds 214–224 displayed selectivity R3

towards the mentioned cancer cells over other tumoral cell
R3 N O
lines and HL-7002 N
non-tumoral cell line. Cl
S lines
displayed selectivity towards the mentioned cancer cells over other tumoral cell and HL-7002
R2 O O R2 O O Cl Pt
non-tumoral cell line.Pt Pt N
R1 R1 Cl
Cl S
R3 R3 N
N R2 O O
R3 R3 O
N N
R2 R2 S Cl
O O O 219-221, Pt
Cl 223
R2 O R2 RO1 O
RO1 O
Pt Pt N
R1 R1 Cl
Cl
214-216 217-218 S
R3 R3 N
N R2 O O
Complex R1 R2 R3 R2 O
R2 O
214 OCH
O 3 O H H O SO Cl S Cl223
219-221,
215 R1 H R 1 Cl Pt
OCH3 H Cl Pt
N N
216 H
214-216 H Cl 217-218
217 H H OH S
218 H F H
219 HR1 HR2 Cl R3 O O O O
Complex O
220 H NMe2 H O
214 OCH3 H H S Cl S Cl
221 H Cl H 222 Pt 224 Pt
215 H OCH3 H Cl Cl
223 H NEt2 H N N
216 H H Cl
217 H H OH S
218 H F H
219 H H Cl O O O O
220 HFigure 70.2 Chemical
NMe H structure of derivatives 214–224.
221 H Cl H 222 224
223 H NEt2 H
However, metal-coumarin complexes constitute an interesting tool not only in the
pharmacological field but also in the70.chemical
Figure Chemicalone. As anofexample
structure of such
derivatives application, we report here
214–224.
a work from Aslkhademi and collaborators, who designed, synthesized ad characterized a new Zn(II)
Figure 70. Chemical structureanofinteresting
derivativestool
complex However, metal-coumarin
with a coumarin-hydrazone complexes
ligandconstitute
(225, Figure 71) [282].214–224.
In not
thisonly in the
case, the pharmacological
complex was
field butasalso
evaluated in the for
a catalyst chemical one. Asofan
the synthesis exampleby
tetrazoles ofmeans
such application, we reportbetween
of 3 + 2 cycloaddition here a work from
a nitrile
However,
Aslkhademi and metal-coumarin
collaborators, whocomplexes
designed, constitute
synthesized anadinteresting
characterized tool
a not Zn(II)
new only complex
in the
and an azide. To this purpose, many experiments were carried out using different nitriles and
pharmacological
with a conditions. field but also
coumarin-hydrazone in the(225,
ligand chemical
Figureone.
71) As an example
[282]. of such
In this complex
case, application, we
the complex report here
reaction In the end, the Zn(II)-coumarin-hydrazone resulted was
to beevaluated
a suitable as a
acatalyst
work from Aslkhademi and collaborators, who designed,
of 3 + amountsynthesized ad characterized a new Zn(II)
catalyst forfor
thethe synthesis
synthesis of tetrazoles
of tetrazoles; thebymost
means
effective 2 cycloaddition
of catalyst between a nitrile
to employ was and
0.05 an azide.
mmol
complex with a many
coumarin-hydrazone ligand (225, Figuredifferent
71) [282]. In this case, the conditions.
complex was
and substrates bearing electron-donating groups reacted better than the ones with electron- In
To this purpose, experiments were carried out using nitriles and reaction
evaluated
the end, theas Zn(II)-coumarin-hydrazone
a catalyst for the synthesis ofcomplex
tetrazolesresulted
by means of 3a+suitable
to be 2 cycloaddition between
catalyst for a nitrile
the synthesis
withdrawing substituents.
and an azide.the
of tetrazoles; Tomost
this effective
purpose,amount
many of experiments were carried
catalyst to employ was 0.05outmmol
usinganddifferent nitriles
substrates and
bearing
reaction conditions. In the end, the Zn(II)-coumarin-hydrazone complex
electron-donating groups reacted better than the ones with electron-withdrawing substituents. resulted to be a suitable
catalyst for the synthesis of tetrazoles; the most effective amount of catalyst to employ was 0.05 mmol
and substrates bearing electron-donating groups reacted better than the ones with electron-
withdrawing substituents.
Figure 71. Chemical structure of Zn(II)-coumarin-hydrazone complex 225.

Figure 71. Chemical structure of Zn(II)-coumarin-hydrazone complex 225.
Finally, it is worth mentioning the application of metal-coumarin complexes as constituents of
sol-gel coatings.
Finally, Lately,
it is worth sol-gels arethe
mentioning gaining attention
application due to their chemical
of metal-coumarin stability,
complexes as homogeneity
constituents ofand
technological
sol-gel coatings.applications (e.g., are
Lately, sol-gels reservoir forattention
gaining the prolonged release
due to theirofchemical
antimicrobial substances).
stability, In 2013,
homogeneity
Jaiswal and co-workers proposed the synthesis of silver-coumarin complexes useful as antibacterial
Figure 71.[283].
agents in sol-gel coatings Chemical structure
These of Zn(II)-coumarin-hydrazone
complexes complex 225.
were based on coumarin-3-carboxylatosilver and its
hydroxylated derivatives in 6, 7 and 8. Antimicrobial activity was displayed even at concentration
Finally,
of 0.3% it isThe
(w/w). worth
mostmentioning the application
active compound showed of significative
metal-coumarin complexes
antibiofilm as constituents
activity at 0.5% andof
sol-gel coatings.
0.7% (w/w). These Lately, sol-gels
results are gaininginattention
are encouraging due to their
the perspective chemical
of using stability, complexes
Ag-coumarin homogeneity as
biomedical coatings.
Int.J.J.J.Mol.
Int.
Int. Mol.Sci.
Mol. Sci.2020,
Sci. 2020,21,
2020, 21,xxxFOR
21, FORPEER
FOR PEERREVIEW
PEER REVIEW
REVIEW 47of
47
47 of83
of 83
83
Int.
Int. J.
J. Mol.
Mol. Sci.
Sci. 2020,
2020, 21,
21, xx FOR
FOR PEER
PEER REVIEW
REVIEW 47
47 of
of 83
83
and
and technological
andtechnological
technological applications
technologicalapplications
applications(e.g.,
applications (e.g.,
(e.g., reservoir
(e.g.,reservoir for
reservoirfor
reservoir forthe
for prolonged
theprolonged
the prolongedrelease
prolonged release
releaseof
release ofantimicrobial
of substances).
antimicrobialsubstances).
substances).
and
and technological applications (e.g.,proposed for the
reservoir the the prolonged release ofof antimicrobial
In
In
In 2013,
2013,
2013, Jaiswal
Jaiswal
Jaiswal and
and
and co-workers
co-workers
co-workers proposed
proposed the
the synthesis
synthesis
synthesis of
of
of silver-coumarin
silver-coumarin
silver-coumarin complexes
complexes
complexes useful
useful as
useful as
as
In
In 2013,
2013, Jaiswal
Jaiswal and
and co-workers
co-workers proposed the
the synthesis
proposed [283]. synthesis of
of silver-coumarin
silver-coumarin complexes
complexes useful
useful asas
antibacterial
antibacterial
antibacterial agents
agents
agents inin
in sol-gel
sol-gel
sol-gel coatings
coatings
coatings [283].
[283]. These
These
These complexes
complexes
complexes were
were
were based
based
based on
on
on coumarin-3-
coumarin-3-
coumarin-3-
antibacterial
antibacterial agents
agents in
in sol-gel
sol-gel coatings
coatings [283].
[283]. These
These complexes
complexes were
were based
based on coumarin-3-
on was
coumarin-3-
carboxylatosilver
carboxylatosilverand
carboxylatosilver and
and its
andits
its hydroxylated
itshydroxylated
hydroxylated derivatives
hydroxylatedderivatives
derivativesin
derivatives in
in6,6,
in 6,7777and
6, and
and8.8.
and Antimicrobial
8.Antimicrobial
8. activity
Antimicrobialactivity
Antimicrobial activitywas
activity displayed
wasdisplayed
was displayed
displayed
Int. J.carboxylatosilver
carboxylatosilver
Mol.
even Sci.
at 2020, 21,
concentrationand
4618 its ofhydroxylated
0.3% (w/w). derivatives
The most in
active 6, 7 and 8.
compound Antimicrobial
showed activity was
significative displayed
antibiofilm 48 of 81
even
even
even atat
at concentration
concentration
concentration of of
of 0.3%
0.3%
0.3% (w/w).
(w/w).
(w/w). The
The
The most
most
most active
active
active compound
compound
compound showed
showed
showed significative
significative
significative antibiofilm
antibiofilm
antibiofilm
even at concentration
activity of 0.3% (w/w). The results
most active compound showed significative antibiofilm
activityatat
activity
activity at 0.5%
0.5% and
at0.5%
0.5% and 0.7%
and 0.7% (w/w).
0.7%(w/w).(w/w). These
(w/w). These results are
Theseresults
results are encouraging
are encouraging in
encouraging in the
in the perspective
the perspective of
perspective of using
of using Ag-
using Ag-
Ag-
activity
coumarin 0.5% and
at complexes and 0.7%
0.7%
as (w/w). These
biomedical These results are
coatings. are encouraging
encouraging in in the
the perspective
perspective of of using
using Ag-
Ag-
coumarin
coumarin
coumarin complexes
complexes
complexes as as
as biomedical
biomedical
biomedical coatings.
coatings.
coatings.
coumarin
Table Table complexes
2 reports
2 reports as
some
some biomedical
ofof the
the coatings. mentioned
coumarins
coumarins mentioned above,
above, summarizing
summarizing the the biological activity, the
Table
Table
Table 2222reports
reports
reports some
some
some of
of
of thecoumarins
the
the coumarinsmentioned
coumarins mentioned
mentioned above,
above, summarizing
summarizing thebiological
the biological
biological activity,
activity,
activity,
the
molecular Table
molecular
target reports
target
and some
and
the of
the
origin. the
origin.coumarins mentioned above, above, summarizing
summarizing the the biological
biological activity,
activity,
the molecular
themolecular
the moleculartarget target
targetandand
andthe the origin.
theorigin.
origin.
the molecular target and the origin.
Table
Table
Table 2.
2.2.Summary
Summary
Summary table.
table.
table.
Table
Table
Table 2. 2. Summary
2. Summary
Summary table. table.
table.
Biological
Biological
Biological
Structure Biological Molecular Target
Structure
Structure Biological Molecular Target Name/Number
Molecular
Molecular Target Name/Number Ref.
Name/Number Ref. Origin
Ref. Origin
Origin
Structure
Structure
Structure Biological
Activity Molecular
Molecular Target
Activity Target Name/Number Ref.
Name/Number Origin
Ref. Origin
Activity
Activity
Activity Target Name/Number Ref. Origin
Activity
Antioxidant
Antioxidant Free
Freeradicals
radicals 33 [30]
[30] Synthetic
Synthetic
Antioxidant
Antioxidant
Antioxidant
Antioxidant
Free
Free radicals
Free radicals
Free radicals
radicals 333 3 [30]
[30] Synthetic
[30]
[30] Synthetic
Synthetic
Synthetic
OH
OH
OH
OH
OH
OH HO OH
HO
HO
HO OH
OH
OH
HO
HO OH
OH
Antioxidant
Antioxidant Free
Freeradicals
radicals 99 [35]
[35] Synthetic
Synthetic
Antioxidant
Antioxidant
Antioxidant
Antioxidant
Free
Free radicals
Free radicals
Free radicals
radicals 999 9 [35]
[35] Synthetic
[35]
[35] Synthetic
Synthetic Synthetic
O O
O
OO OO
O
O O OO
OO
O O
O
O OO
OO OO
O
O
OO
O
O O
O
OO
O
O
Antioxidant
Antioxidant
Antioxidant Free
Freeradicals
Free radicals
radicals 10d
10d
10d [36]
[36] Synthetic
[36] Synthetic
Synthetic
O
OO Antioxidant
Antioxidant
Antioxidant Free
Free radicals
Free radicals
radicals 10d
10d 10d [36]
[36] Synthetic
[36]
Synthetic Synthetic
O OO OO O
O
OO O OO
HO
O O OO
O HO
OHO O O O
HO
HO N
HO N
NN
N
N
Antioxidant
Antioxidant
Antioxidant Free
Freeradicals
Free radicals
radicals 15
15
15 [38]
[38] Synthetic
[38] Synthetic
Synthetic
Antioxidant
Antioxidant Free
Free radicals
radicals 15
15 [38]
[38] Synthetic
Synthetic
Antioxidant Free radicals 15 [38] Synthetic
Antiprolifera-
Antiprolifera-
Antiprolifera-
Antiprolifera-
Antiprolifera- DNA, 18
DNA,chromatin
DNA, chromatin
chromatin 18
18
18
[45]
[45] Natural
[45] Natural
Natural
tive DNA, chromatin
DNA,DNA,
chromatin 18 [45]
[45] Natural
Natural
tive
tive
Antiprolifera-tive chromatin 18 [45] Natural
tive
tive
Antiprolifera-
Antiprolifera-
Antiprolifera-
Antiprolifera-
Antiprolifera- Topoisomerase 111 20 [47] Synthetic
Antiprolifera-tiveTopoisomerase
Topoisomerase
Topoisomerase 1 20
20 20 [47]
[47] Synthetic
Synthetic
[47] Synthetic
tive Topoisomerase
Topoisomerase 11 20
20 [47]
[47] Synthetic
Synthetic
tive
tive
tive
tive
Antiprolifera- Antiprolifera- Tumor-suppressor

Antiprolifera- Tumor-suppressor
Tumor-suppressor
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEWAntiprolifera- Tumor-suppressor 48 of 83
Tumor-suppressor 21
Antiprolifera- Tumor-suppressor 21
21 [49]
[49]
[49] Synthetic
Synthetic
Synthetic
Antiprolifera-tive
tive protein p53 21
21 21 [49]
[49] [49]
Synthetic
Synthetic Synthetic
tive
tive protein
protein p53 p53
p53
protein
tive
tive protein
protein p53
p53
Anticancer 1 22a-l [50] Synthetic
Anticancer
Anticancer 22a-l 22a-l [50] Synthetic
[50] Synthetic
O N
N O
Br O Anticancer STAT3STAT3 23 [55] Synthetic
O N Anticancer 23 [55] Synthetic
N
S O N O
Br O Anticancer STAT3 23 [55] Synthetic
O O
N
S O Carbonic
O O
Anticancer Anhydrase IX and 24 [70] Natural
Carbonic
XII
Anticancer Anhydrase IX and 24 [70] Natural
XII
HO O O
N
N
R HO O O
Carbonic
N
N Anticancer Anhydrase IX and 26–37 [71] Synthetic
26: R 33:
HO O O F Carbonic
27: 34:
Anticancer 22a-l [50] Synthetic
O N
OO NN
Int. J. Mol. Sci. 2020,
O 21, 4618 N 49 of 81
N
ON O N
Br N OO
BrBr N O O
OO
Anticancer
Anticancer
Anticancer STAT3
STAT3
STAT3 23
23
23 [55]
[55]
[55] Synthetic
Synthetic
Synthetic
Br N O Anticancer STAT3 23 [55] Synthetic
NN N O
Br N O Anticancer STAT3 23 [55] Synthetic
S O
NO
SSO O Table 2. Cont.
S OO
O
OOO
OSO O
O O Carbonic
Molecular
Carbonic
Carbonic
Structure Biological Activity Carbonic Name/Number Ref. Origin
CarbonicTarget
Anticancer Anhydrase
Anticancer AnhydraseIX IX and
IXand
and 24
24
24 [70] Natural
[70] Natural
[70] Natural
Anticancer AnhydraseCarbonic
IX and 24 [70] Natural
Anticancer Anhydrase XII
XII
XII IX and 24 [70] Natural
XII IX 24 [70] Natural
XII and XII
HO
HO O O
HO OO OO
HO O O
N
NN
HO N O O
N
R NN
RRN N
R Carbonic
Carbonic
Carbonic
N Carbonic
R
26: 33: Anticancer
Anticancer Carbonic
Anhydrase
AnhydraseCarbonic
IX and
IXand
IX and 26–37
26–37
26–37 [71] Synthetic
[71] Synthetic
[71] Synthetic
26:26:
26: HO HO
HO
O O
OO OO
33:33:
33: F
FF
Anticancer
Anticancer Anhydrase IX and IX
Anhydrase 26–37 26–37 [71] Synthetic
[71] Synthetic
27:HO
27:27:
26: CH
O O
CH
CH 3
3 3
34:
34:
33:
34:
F Anticancer Anhydrase
XII IX and
XIIand XII
XII 26–37 [71] Synthetic
Br
27:
28: HO
28:28:
CH
CH3O
CHCH2CH3
2CH2CH
O
3 3
34:
FBr
Br
Br
XII
CH CH 35:
28:
27:
29:
29:29:
CH
CH
CH
2 23CH
CH2CH
3 2OH
2CH 2OH
2OH
34:
35:35:
35: Br
XII
28:
29: CH
CH2 CHCH2 OH
30 CH22CHCH 3 2
3030 CHCH 2CHCH
2CHCH 2 2 35:
36
3029: CH2CHCH
CH 2CH2OH 2 : 36
36 F
31: : :
36 FF
31:31: CH2CHCH2 : F
30
31:
36 O
32: :3 3 F OO
32:
32: 37:
31: OO
Cl 37: 7:
32: OO
ClCl 7: OO
32:
Cl 3
7: O
Cl
NNHR
NNHR
NNHR
NNHR
S.S.aureus,
S. aureus, E.
aureus,E. coli,
E.coli, P.
coli,P.
P.
NNHR S. aureus, E. coli,
S. aureus, P. coli, P.
E.
Antibacterial/a
Antibacterial/a
Antibacterial/a
HO OO O OO Antibacterial/a aeruginosa,
S. aureus, E.
A. coli, P.
niger,
aeruginosa, 38,39 [79] Synthetic
HO
HO O aeruginosa,A.
aeruginosa, niger,A. 38,39
A.niger, 38,39
38,39 [79] Synthetic
[79] Synthetic
HO O O Antibacterial/a
ntifungal aeruginosa,
Antibacterial/antifungal A. niger, 38,39 [79] Synthetic
[79] Synthetic
ntifungal
ntifungal niger,A.A.niger,
flavus, C. 38,39
HO NNHR O
NNHR
NNHR O ntifungal aeruginosa,
A. flavus, C. albicans [79] Synthetic
NNHR A.A.flavus,
flavus,C.
C.albicans
albicans
albicans
ntifungal A. flavus, C. albicans
R=-
R=-CONH
CONH or
NNHR
or -CSNH
-CSNH
R=- CONH or -CSNH 2
2 A. flavus, C. albicans
R=- CONH22 2or -CSNH22 2
R=- CONH2 or -CSNH2
S.S.aureusus,
S. aureusus,
aureusus,
Antibacterial S. aureusus, 40,41a–e
S. aureusus, 40,41a–e [80] Synthetic
Antibacterial
Antibacterial
Antibacterial 40,41a–e [80]
[80]
40,41a–e[80] Synthetic
Synthetic
[80] Synthetic
Antibacterial S. aureusus,
E.faecalis, E. coli
E.faecalis, 40,41a–e Synthetic
E.faecalis,
E.faecalis, coliE. coli
E.coli
E.
Antibacterial E.faecalis, E. coli 40,41a–e [80] Synthetic
E.faecalis, E. coli
S.S.aureus
S. aureus
aureus DNA
DNADNA
DNA
S. aureus
Antibacterial S. aureus DNA
Antibacterial
Antibacterial
Antibacterial 42–49
42–49 42–49
42–49 [82]
[82]
[82] Synthetic
[82]
Synthetic
Synthetic Synthetic
Antibacterial S. aureus
gyrasegyrase
DNA 42–49 [82] Synthetic
gyrase
gyrase
Antibacterial gyrase 42–49 [82] Synthetic
Int.J. J.Mol.
Int. Mol.Sci.
Sci.2020,
2020,21,
21,x xFOR
FORPEER
PEERREVIEW
REVIEW gyrase 4949ofof8383
Antibacterial
Antibacterial S.S.aureus
aureus 50,51
50,51 [83] Synthetic
Antibacterial S. aureus 50,51 [83] Synthetic
[83] Synthetic
Modulatorofof
Modulator isopimpinellin
isopimpinellin
Modulator of n.g. n.g. isopimpinellin
[85] Natural
Natural
n.g. [85] [85] Natural
antibiotics
antibiotics
antibiotics imperatorin
imperatorin
imperatorin
6’,7’-
6’,7’-
S.S.aureus,
aureus,S.S.
dihydroxyberga
dihydroxyberga
epidermidis,
epidermidis,
mottin,
mottin,
Antibacterial P.aeruginosa,
Antibacterial P.aeruginosa,E.E. [86] Natural
[86] Natural
peucedanin,
peucedanin,
cloacae,K.K.
cloacae,
officinali
officinali
Pneumoniae,E.E.coli
Pneumoniae, coli
isobutyrate
isobutyrate
HOOC
HOOC
O
O N
N
N
N
NH N
H
Et N O O
Et2N2 O O
Cl
Cl
EtOOC
EtOOC
O
O S.S.aureus,
aureus,L.L.
Int. J. Mol. Sci. 2020, 21, 4618 50 of 81

Modulator of isopimpinellin
n.g. [85] Natural
antibioticsof
Modulator imperatorin
isopimpinellin
Cont.
Table 2.n.g. [85] Natural
antibiotics imperatorin
Molecular
Structure Biological Activity Name/Number Ref. Origin
Target
6’,7’-
S. aureus, S.
dihydroxyberga
6’,7’-
epidermidis,
S. aureus, S.
mottin,
dihydroxyberga
Antibacterial S. aureus,
P.aeruginosa,
epidermidis, E. S. [86] Natural
epidermidis, P. peucedanin, 0 0
mottin, 6 ,7 -
Antibacterial cloacae, K.
aeruginosa, E. dihydroxybergamottin,
Antibacterial P.aeruginosa, E. officinali
[86] Natural
[86] Natural
cloacae,
Pneumoniae, E. K. peucedanin,
coli peucedanin,
cloacae, K.
Pneumoniae, E. isobutyrate
officinali isobutyrate
officinali
coli
Pneumoniae, E. coli
isobutyrate
HOOC
O
N
N
HOOC
HOOC N
OH
O
Et2N O O N
N
N
N N
N
H
H
Et22N
Et N O
O O
O Cl
EtOOC
O Cl
Cl
S. aureus, L.
N
N
EtOOC N monocytogenes,
OH S. aureus, L. E. L.
S. aureus,
Et2N O O N
N N Antibacterial coli, monocytogenes,
Salmonella - E. 52–54 [87] Synthetic
H monocytogenes,coli,E.
Et22N O O Cl Antibacterial inhibitors of Topo 52–54 [87] Synthetic
Antibacterial coli, Salmonella—inhibitors
Salmonella - 52–54 [87] Synthetic
of Topo II and
HOOC Cl II and Topo
inhibitors Topo IV
of Topo
O IV
Cl N
N
HOOC
HOOC N II and Topo IV
OH
O
Cl O O N
N
Cl N
N
N N
H
H
O
O O
O Cl
Cl
Cl
C. albicans -P450
C. albicans -P450
cytochrome
cytochrome
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW Antifungal C. albicans -P450 55–59 50 of[94]
83 Synthetic
Antifungal lanosterol 55–59 [94] Synthetic
lanosterol 14α-
14α-demethylase
cytochrome
OH
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW Antifungal 55–59 [94] Synthetic
50 of 83
O demethylase
C. albicans14α-
lanosterol -
R
OH inhibition
demethylase
OH
O C. albicans -
R ergosterolC.
N Antifungal inhibition 60,61 [95] Synthetic
OH albicans—inhibition
biosynthesis by
O O ergosterol
ergosterol
N Antifungal bindingbiosynthesis
Antifungal lanosterol by 60,61 60,61
[95] [95]
Synthetic Synthetic
biosynthesis by
binding
O O 14a-demethylase
R =Me or Et lanosterol
binding14a-demethylase
lanosterol
Several Candida
14a-demethylase
R =Me or Et
strains - mode of
SeveralSeveral Candida
Candida
actionstrains—mode
involving of
strainsaction
- mode of
involving
Antifungal CYP51 and 62 [96] Synthetic
Antifungal CYP51 and 62 [96] Synthetic
action involving
additional
additional
Antifungal CYP51 unidentified
and 62 [96] Synthetic
unidentified
mechanism
additional
mechanism
unidentified
mechanism
marmesin
senesioate,
Antifungal C. albicans marmesin [97] Natural
suberosin,
senesioate,
Antifungal C. albicans crenulatin [97] Natural
suberosin,
crenulatin
C. albicans -
Antifungal coumarin [100] Natural
antibiofilm action
C. albicans -
antibiofilm action
R =Me or Et Several Candida
14a-demethylase
14a-demethylase
RR=Me
=MeororEt
Et
Several
strains Candida
- mode of
Several
Several Candida
Candida
strainsinvolving
action - mode of
strains
strains - -mode
modeofof
Antifungal action
CYP51 involving
and 62 [96] Synthetic
Int. J. Mol. Sci. 2020, 21, 4618 action
action involving
involving 51 of 81
Antifungal CYP51
additional and 62 [96] Synthetic
Antifungal
Antifungal CYP51
CYP51 and
and 62
62 [96]
[96] Synthetic
Synthetic
additional
unidentified
additional
additional
2. Cont.
Tablemechanism
unidentified
unidentified
unidentified
mechanism Molecular
Structure Biological Activitymechanism
mechanism Name/Number Ref. Origin
Target
marmesin
marmesin
senesioate,
Antifungal C. albicans marmesin
marmesin [97] Natural
senesioate,
suberosin,
Antifungal C. albicans marmesin
senesioate, [97] Natural
senesioate,
suberosin,
Antifungal
Antifungal C.
C.albicans
albicans crenulatin
senesioate,
[97]
[97] Natural
Natural
Antifungal C. albicans [97] Natural
suberosin,
suberosin,
suberosin,
crenulatin
crenulatin
crenulatin
crenulatin
C. albicans -
C. albicans
antibiofilm action-
Antifungal C. coumarin [100] Natural
C.albicans
albicans
antibiofilm
- -C.
action
Antifungal
Antifungal coumarin
coumarin [100]
[100] Natural
Natural
albicans—antibiofilm
Antifungal antibiofilm coumarin [100] Natural
antibiofilmaction
action
action
F. oxysporum, A.
Antifungal 63–68 [102] Synthetic
F. oxysporum,
flavus, A. nigerA.
Antifungal F. oxysporum, A. 63–68
Antifungal F.F.oxysporum,
oxysporum, A.
A. 63–68 [102] Synthetic
[102] Synthetic
Antifungal flavus, A. niger
flavus, A. niger 63–68 [102] Synthetic
Antifungal 63–68 [102] Synthetic
flavus,
flavus,A.A.niger
niger
Anti-HIV reverse
Antiviral Anti-HIV reverse 69–71
Antiviral 69–71[113] Natural
[113] Natural
Anti-HIV reverse
transcriptase
transcriptase
Antiviral Anti-HIV reverse 69–71 [113] Natural
Anti-HIV reverse
transcriptase
HO Antiviral
Antiviral 69–71
69–71 [113]
[113] Natural
Natural
OH
transcriptase
transcriptase
HO
Int.
Int.J.J.Mol.
Mol.Sci. OH 21,
Sci.2020,
2020, 21,xxFOR
FORPEER
PEERREVIEW
REVIEW 51
51of
of83
83
HO
HO HO OH O O
OH
HO HO O O
HO
OO
HO
HO OO OO
OO OO OO
OO HIV
HIVreverse
reverse
Antiviral
Antiviral HIV reverse 72–74
72–7472–74 [116] Natural
[116] [116]
Natural
Antiviral Natural
transcriptase
transcriptase
transcriptase
OO OO OO
RR
OO
HH
RR11 NN
NN
HH
OO OH
OH
OO OO
Antiviral
Antiviral HIV1-IN
HIV1-IN 75–78
75–7875–78 [117]
[117] Synthetic
Synthetic
RR22 Antiviral HIV1-IN [117] Synthetic
75
75 RR11Br
Br RR22HH RRHH
76
76 RR11Cl
Cl RR22HH RRHH
77
77 RR11HH RR22OMe
OMe RR5'-Cl
5'-Cl
78
78 RR11HH RR22OEt
OEt RR5'-Cl
5'-Cl
ClCl
ClCl
SS
NH
NH
NN
OO OO
Antiviral
Antiviral H1N1
H1N1virus
virus 79–82
79–82 [125]
[125] Synthetic
Synthetic
R
O
H
R1 N
N
H R
O O OH
O O H
R1 N Antiviral HIV1-IN 75–78 [117] Synthetic
R2 N R
H
O O OH
Int. J. 75 Sci.
Mol. R1 Br O RO
2020, 221, H RH
H 4618 52 of 81
R1 76 R Cl R H N R H Antiviral HIV1-IN 75–78 [117] Synthetic
R2 1 2 N
77 R1 H R2 OMeH R 5'-Cl
O OH
78
75 R HO
11 Br R O OEt
22 H RRH5'-Cl
Antiviral HIV1-IN 75–78 [117] Synthetic
76 RR21 Cl R2 H RH
77 R1 H R2 OMe R 5'-Cl Table 2. Cont.
75 R1 H
78 Br R2 OEt
H RRH5'-Cl
76 R1 Cl R2 H RH
77 R1 H Structure
R2 OMe R 5'-Cl
Molecular
Biological Activity Name/Number Ref. Origin
78 R1 H R2 OEt RCl5'-Cl Target
Cl
S
NH Cl
N
O O S Cl
NH Cl
N
Antiviral H1N1 virus 79–82 [125] Synthetic
Cl
O O S
NH
N
Antiviral H1N1 virus 79–82 [125] Synthetic
O O
Antiviral
Antiviral H1N1H1N1
virus virus [125]
79–82 79–82 [125] Synthetic Synthetic
Antiviral Influenza A virus 83 [128] Synthetic

Antiviral Influenza
Influenza A virus 83 [128] Synthetic
A/PR8/H1N1
Influenza
Influenza
A/PR8/H1N1
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW virusesviruses—probably
– probably 52 of 83
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW Antiviral A/PR8/H1N1 84–89 [129] Synthetic
52 of 83
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW Antiviral could could
affect affect
Influenza 84–89 [129]
52 of 83 Synthetic
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW
viruses – intracellular
probably 52 of 83
Antiviral A/PR8/H1N1
intracellular redox-
redox-sensitive 84–89 [129] Synthetic
could affect
pathways
viruses – pathways
sensitive probably
Antiviral intracellular redox- 84–89 [129] Synthetic
Antiviral Hepatitis C virus
could affect 90–92 [130] Synthetic
Antiviral Hepatitis
sensitive C virus 90–92 [130] Synthetic
Antiviral Hepatitispathways
C virus 90–92 [130] Synthetic
intracellular redox-
Antiviral Hepatitis C virus 90–92 [130] Synthetic
Antiviral
AntiviralHepatitis
sensitive virus C virus90–92 90–92
Cpathways
Hepatitis [130] Synthetic
[130] Synthetic
Antiviral Hepatitis B virus esculetin [131] Natural

AntiviralHepatitisHepatitis
Antiviral B virus B virus
esculetin esculetin [131]
[131] Natural Natural
Antiinflamma-
Antiinflamma- COX2 93–100 [143] Synthetic
Antiinflamma-
tory COX2 93–100 [143] Synthetic
tory
Antiinflamma-tory
tory
Antiinflamma-
Antiinflamma- 5-LOX 101,102 [146] Synthetic
Antiinflamma-
tory
Antiinflamma-tory 5-LOX 5-LOX 101,102 101,102 [146]
[146] Synthetic Synthetic
tory
tory
tory 5-LOX 101,102 [146] Synthetic
tory
Antiinflamma-
Antiinflamma- NO production 103 [147] Natural
Antiinflamma-tory
Antiinflamma-
tory NO production 103
NO production [147]
103[147] Natural Natural
tory
tory
tory NO production 103 [147] Natural
tory
NF-kB and
Antiinflamma- NF-kB and
p38/MAPK osthole [150] Natural
Antiinflamma-
tory p38/MAPK
NF-kB and osthole [150] Natural
tory
Antiinflamma- p38/MAPK
signaling
NF-kB and pathways osthole [150] Natural
tory
Antiinflamma- signaling pathways
tory signaling
p38/MAPKpathways osthole [150] Natural
tory signaling pathways
signaling pathways
Antiinflamma- NF-kB signaling
Antiinflamma- NF-kB signaling 104 [151] Synthetic
Antiinflamma-
tory NF-kBpathways
signaling 104 [151] Synthetic
tory
Antiinflamma- pathways
NF-kB signaling 104 [151] Synthetic
tory pathways
tory
Antiinflamma-
tory NO production 103 [147] Natural
tory
Antiinflamma-
Int. J. Mol. Sci. 2020, 21, 4618 NO production 103 [147] Natural 53 of 81
tory
Antiinflamma-
NO production 103 [147] Natural
tory NF-kB and
NF-kB
Antiinflamma-Table 2. Cont. and
Antiinflamma- p38/MAPK osthole [150] Natural
tory p38/MAPK osthole [150] Natural
tory NF-kB
signaling Molecular
and
pathways
Antiinflamma- signaling pathways
Target
tory NF-kB and
signaling pathways
p38/MAPK
p38/MAPK osthole
Antiinflamma-tory
Antiinflamma- NF-kB signaling osthole [150] Natural
[150] Natural
tory
Antiinflamma- signaling
tory signaling pathways
pathways
pathways 104 [151] Synthetic
tory pathways
Antiinflamma-tory
tory pathways 104 [151] Synthetic
pathways
Anti- 104 [151] Synthetic
Anti-
tory AChE, pathways
BuChE,
Alzhemer’s AChE, BuChE, 105 [162] Synthetic
Alzhemer’s
Anti-Alzhemer’s AChE, BuChE,
BACE1 105 [162] Synthetic
Anti- BACE1 105 [162] Synthetic
disease
disease BACE1
disease AChE, BuChE,
Alzhemer’s 105 [162] Synthetic
Anti- BACE1
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW Anti- AChE,
AChE,BuChE,
BuChE, 53 of 83
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW disease Anti- AChE,AChE,
BuChE, 53 of 83
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW Alzhemer’s BuChE, 105 53 of 83
[162] Synthetic
Anti-Alzhemer’s BACE1,
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW Alzhemer’s metal
53 of 83
Anti-
Alzhemer’s BACE1, BACE1,
metal metal 107 107 [163] Synthetic [163] Synthetic
Int.
Int. J.J. Mol.
Mol. Sci.
Sci. 2020,
2020, 21,
O
21, xxOFOR
O
FOR PEER
PEER REVIEW
REVIEW
disease
Anti-
disease 53
53 of
of 83
83
Anti- AChE,
AChE, chelation
BuChE,
BuChE,
N N disease chelation
AChE, BuChE,
HN N
O Alzhemer’s
disease
Anti- AChE, BuChE,
chelation 108 [163] Synthetic
Br O O ONO H N
O Alzhemer’s 108 [163] Synthetic
Br N O O OH
O Alzhemer’s
Anti- BACE1,BACE1
AChE, metal
BuChE, 108
107 [163] Synthetic
Br NN N
N N N O O O
O
OO N N Anti-
Anti- BACE1
AChE, BuChE,
H disease
Alzhemer’s AChE,BACE1 108 [163] Synthetic
N N N N O disease AChE, BuChE,
BuChE,
Br N O O N
N
O
H
H
N
N disease
Alzhemer’s
Anti-Alzhemer’s chelation
BACE1
AChE, BuChE, 108
108 [163] Synthetic
N O
H O
O
O Alzhemer’s
Alzhemer’s BACE1, metal 107 [163]
[163] Synthetic
Synthetic
Br
Br
Br N
N
N OO O
O
O O
OO disease BACE1
N
N Anti-
disease BACE1
N
NN NN Anti-
disease
disease
Anti-
disease chelation
Alzhemer’s
Anti- AChE, BuChE 111a [166] Synthetic
Alzhemer’s AChE, BuChE 111a [166] Synthetic
Alzhemer’s
Anti- AChE, BuChE 111a [166] Synthetic
Anti-
disease
Alzhemer’s
disease AChE, BuChE 111a [166] Synthetic
disease
Alzhemer’s AChE, BuChE 111a
111a 111a [166]
Anti-Alzhemer’s
Alzhemer’s AChE, BuChE
AChE, BuChE [166] Synthetic
Synthetic
[166] Synthetic
disease
disease
disease
Anti-
disease
Anti-
Anti- AChE, BuChE,
AChE, BuChE,
Alzhemer’s
Anti- AChE, BuChE, 117 [167] Synthetic
Alzhemer’s 117 [167] Synthetic
Alzhemer’s
Anti- MAO-B
AChE, BuChE, 117 [167] Synthetic
Anti- MAO-B
disease MAO-B
AChE,
Alzhemer’s
BuChE, 117 [167] Synthetic
disease
Alzhemer’s 117
Anti-Alzhemer’s
Alzhemer’s MAO-B
AChE, BuChE, 117 117 [167][167] Synthetic
Synthetic
[167] Synthetic
disease
disease MAO-B
MAO-B MAO-B
Anti-
Anti-
Anti- AChE, BuChE,
Alzhemer’s
Anti- MAO-B,
AChE,β-amyloid
Alzhemer’s MAO-B, β-amyloid 136 [169] Synthetic
Alzhemer’s
Anti- MAO-B,
AChE, β-amyloid
Anti-
AChE,
aggregation BuChE,
Alzhemer’s
disease MAO-B, β-amyloid
aggregation 136 [169] Synthetic
disease
Anti-Alzhemer’s
Alzhemer’s aggregation
MAO-B, 136
Alzhemer’s MAO-B,
MAO-B, β-amyloid
β-amyloid 136 136 [169][169] Synthetic
Synthetic
[169] Synthetic
disease
disease aggregation
β-amyloid
disease
disease aggregation
aggregation
aggregation
Anticonvulsant GABAergic system 148 [180] Synthetic
Anticonvulsant GABAergic GABAergic
system 148 [180] Synthetic
Anticonvulsant 148 [180] Synthetic
Anticonvulsant GABAergicsystem
Anticonvulsant GABAergic system
system 148
148 [180]
[180] Synthetic
Synthetic
Anticonvulsant BDZ receptor 152d [183] Synthetic

Anticonvulsant
Anticonvulsant BDZ receptor
BDZ receptor 152d 152d [183] Synthetic
[183] Synthetic
Anticonvulsant
Anticonvulsant BDZ receptor
BDZ receptor 152d
152d [183] Synthetic
[183] Synthetic
Tecarfarin [195–
Tecarfarin [195–
Anticoagulant VKOR inhibitor Tecarfarin [195–
Tecarfarin
Anticoagulant VKOR
Anticoagulant VKOR inhibitor
VKOR inhibitor (ATI-5923) [195–198] Synthetic
Anticoagulant inhibitor (ATI-5923)198]
Tecarfarin
(ATI-5923) [195– Synthetic
198] Synthetic
Anticoagulant VKOR inhibitor (ATI-5923)
Tecarfarin 198] Synthetic
[195–
Tecarfarin [195–
Anticoagulant VKOR inhibitor (ATI-5923) 198] Synthetic
(ATI-5923)
(ATI-5923) 198]
198] Synthetic
Synthetic
Anticoagulant n.g. 154,155 [199] Synthetic

Anticoagulant
Anticoagulant n.g. n.g. [199]
154,155154,155[199] Synthetic Synthetic
Anticoagulant
Anticoagulant n.g.
n.g. 154,155
154,155 [199]
[199] Synthetic
Synthetic
murine VKORC1
murine VKORC1 156
Anticoagulant murine VKORC1 156 [200] Synthetic
Anticoagulant [200] Synthetic
Anticoagulant inhibitor
murine VKORC1 156 [200] Synthetic
inhibitor
inhibitor
Anticoagulant murine
murine VKORC1
VKORC1 156 [200] Synthetic
Anticoagulant
Anticoagulant inhibitor 156
156 [200]
[200] Synthetic
Synthetic
inhibitor
inhibitor
(ATI-5923) 198] Synthetic
Int. J. Mol. Sci. 2020, 21, 4618 54 of 81

Table 2. Cont.
Molecular
Target
Int.Int.
Int.J.J.
Int.
Int. J.J.
J.Mol. Mol.
Mol.
Mol.
Mol.Sci.Sci.
Sci.
Sci.
Sci. 2020,
2020,
2020,
2020,
2020, 21,21,
21,
21,
21, x
x xxFORFOR
xFOR
FOR
FOR PEER
PEER
PEER
PEER
PEER REVIEW
REVIEW
REVIEW
REVIEW
REVIEW murine VKORC1 545454
54
54 of
of
of of
of
838383
83
83
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW Anticoagulant murine VKORC1 156 [200] Synthetic
54 of 83
Anticoagulant inhibitor 156 [200] Synthetic
inhibitor
Anticoagulant
Anticoagulant
Anticoagulant
Anticoagulant n.g.
n.g.
n.g.
n.g. 157
157
157
157 [203] Natural
[203]
[203]
[203] Natural
Natural
Natural
Anticoagulant n.g. 157 [203] Natural
Anticoagulant n.g. 157 [203] Natural
Antidiabetic α-glucosidases
Antidiabetic
α-glucosidases
α-glucosidases 158d,e
158d,e
158d,e [207] Synthetic
[207]
[207] Synthetic
Synthetic
Antidiabetic
Antidiabetic α-glucosidases 158d,e 158d,e[207] Synthetic
[207] Synthetic
Antidiabetic α-glucosidases 158d,e [207] Synthetic
d:d:
R
d:d:
d:R
R =11R=
1R 1=
=
=H,
1 H,H,
H,
H,RR
R2R=22R== 2=
=
2NEt NEt
NEt
NEt NEt
2,22R 3,R
,2, ,2R
R 33R
=H=H3=H
3=H
=H
Re:
e:e:
e:
e:
d:
R
1R
RR=11R= =OCH
1OCH
=
1OCH
1==OCH
OCH
H, R3,33R
2=
2,R
,3, ,3R
R =22R
NEt
==
=H,
2 =
2H,
H,H,
2, R
RH,R
3R
R 3R
=H
33
=H3=H
3=H
=H
=H
e: R1= OCH3, R2= H, R3=H
Antidiabetic
Antidiabetic
Antidiabetic
α-glucosidases
159d,e
159d,e
Antidiabetic α-glucosidases [207]Synthetic
[207]
[207]
159d,e[207] Synthetic Synthetic
Synthetic
Synthetic
[207]
Antidiabetic α-glucosidases 159d,e [207] Synthetic
Antidiabetic
Antidiabetic
α-glucosidases
α-glucosidases 165
165
165
165 [214] Synthetic
[214]
[214] Synthetic
Synthetic
Antidiabetic α-glucosidases 165 [214] Synthetic
[214] Synthetic
Antidiabetic α-glucosidases 165 [214] Synthetic
ClCl
Cl Cl
Cl
NNN
NN NNN
NN
Cl NNN
N
N NN
N
OOO
OO
Antidiabetic
α-glucosidases
α-glucosidases 168c
168c
168c [215] Synthetic
[215]
[215] Synthetic
Synthetic
O
Antidiabetic
Antidiabetic α-glucosidases 168c 168c[215] Synthetic
[215] Synthetic
Antidiabetic α-glucosidases 168c [215] Synthetic
OH
OH
OHOH
OH OH
OH OH
OH
OH
OH OH
OOO OO
OO OOOO OO
OO
OO OOO
O OO O
Antidiabetic Insulin
Antidiabetic
Antidiabetic Insulin
Insulin
Insulin release
release
release 169–171 [217]
169–171
169–171 [217]Synthetic
[217]
[217] Synthetic
Synthetic
Synthetic
Antidiabetic
Antidiabetic release
Insulin release 169–171169–171 [217] Synthetic
Antidiabetic Insulin release 169–171 [217] Synthetic
R1R
RR11R1 1R2R22R
RR 2 2R3R33R
RR R
RR
3 3R4 44R R
RR
4 4R5 55R55
169
169 169
169
169 HH HHHSMe SMeSMeH H
SMe
SMe HHHOMe OMe OMe
OMe
OMe OMe
OMe OMe
OMe
OMe
170
170 170
170
170 HHRHH 1HSMe RSMe
SMe
SMe
SMe 2 HRH
HH3 H HR H
HH4 HOH ROH
OH
OH 5OH
171 171
169
171
171
171 HH HHHSMe
H SMe
SMeSMe
SMe
SMe OMe
OMe HOMe
OMe
OMe OHOMe
OH
OHOHOHHOMe
HHHH
170 H SMe H H OH
171 H SMe OMe OH H
Int. J. Mol. Sci. 2020, 21, 4618 55 of 81
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW Table 2. Cont. 55 of 83
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW Molecular 55 of 83
Structure Biological Activity Name/Number 55 of 83
Ref. Origin
Target
Photocleavable
Photocleavable [231,
Photocleavable
protecting - 172–174 [231, Synthetic
Photocleavable [231,
Photocleavable
protecting - 172–174 232] Synthetic
protecting - 172–174 [231, Synthetic
groups 172–174 [231,
232]
protecting
Photocleavable - 232]
172–174 172–174 Synthetic
protecting
groups - - Synthetic
[231,232] Synthetic
protecting
groups groups 232]
232]
groups
groups
Photocleavable
Photocleavable
Photocleavable
protecting - 175 [224] Synthetic
Photocleavable
Photocleavable
protecting - 175 [224] Synthetic
protecting
Photocleavable
group - 175 [224] Synthetic
protecting - - 175 175[224] Synthetic
[224] Synthetic
protecting
group
protecting group - 175 [224] Synthetic
group
group
group
Photocleavable
Photocleavable
Photocleavable
protecting - 176–178 [233] Synthetic
Photocleavable
Photocleavable
protecting
Photocleavable - 176–178 [233] Synthetic
protecting
groups groups - - 176–178 176–178
[233] Synthetic
[233] Synthetic
protecting
protecting - 176–178 [233] Synthetic
protecting
groups - 176–178 [233] Synthetic
groups
groups
groups
GHS (in the

GHS (in the [236,
Ionic probe GHS (in
presence the
ofGHS
Cu2+)(in the 179 [236, Syntehtic
GHS (in the
IonicIonic
probe GHS (in
probepresence Cu2+) of Cu2+ )179
ofthe
presence 179[236,
237]
[236, Syntehtic
[236,237] Syntehtic
Ionic probe presence
and of
ClO Cu
-
and
2+)
2+ClO
− 179 [236,
237] Syntehtic
Ionic probe presence of Cu- 2+)) 179 237] Syntehtic
Ionic probe presence of Cu
and ClO 179 Syntehtic
and ClO-- 237]
237]
and ClO-
and ClO
IonicIonic
probeprobe ClO- ClO− 180 180[238] Synthetic
[238] Synthetic
Ionic probe ClO- 180 [238] Synthetic
Ionic probe ClO-- 180 [238] Synthetic
Ionic probe ClO- 180 [238] Synthetic
Ionic probe ClO 180 [238] Synthetic
IonicIonic
probeprobe ClO-, CuClO
2+ −
, Cu2+ 181 181[239] Synthetic
[239] Synthetic
Ionic probe ClO-, Cu2+ 181 [239] Synthetic
Ionic probe ClO--, Cu2+ 181 [239] Synthetic
Ionic probe ClO , Cu2+ 181 [239] Synthetic
Ionic probe ClO-, Cu2+ 181 [239] Synthetic
IonicIonic
probeprobe ClO- ClO− 182–184 182–184
[240] Synthetic
[240] Synthetic
Ionic probe Cu2+ 185 [241] Synthetic


Ionic probe
Ionic probe ClO---
ClO 182–184
182–184 [240]
[240] Synthetic
Synthetic
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW Ionic probe ClO- 182–184 [240] Synthetic
56 of 83
Ionic probe ClO- 182–184 [240] Synthetic
Int. J. Mol. Sci. 2020, 21, 4618 56 of 81

Ionic probe
Ionic probe Table Cu2+
2.
Cu
2+
2+
Cont. 185
185 [241] Synthetic
[241] Synthetic
Ionic probe Cu 2+ 185 [241] Synthetic
Ionic probe Cu2+ Molecular 185 [241] Synthetic
Target
IonicIonic
Ionic probe
probeprobe Cu2+
Cu
2+
2+ Cu2+ 185
186 [241]
185[242] Synthetic
[241]
Synthetic Synthetic
IonicIonic
probeprobe Cu2+ Cu2+ 186 186[242] Synthetic
[242] Synthetic
Ionic probe
Ionic probe Cu2+
Cu
2+
2+
2+ 186
187 [242]
[243] Synthetic
Synthetic
Ionic probe Cu 187 [243] Synthetic
Ionic probe Cu 2+ 187 [243] Synthetic
IonicIonic
probeprobe Cu2+ Cu2+ 187 187[243] Synthetic
[243] Synthetic

Cu2+
Cu
2+, Al3+
2+
, Al3+,, Arg,
3+ Arg,
Ionic probe
probe Cu2+, Cu
Al3+2+, Arg, 187
2+ , Al3+ , Arg, 188 [243] Synthetic
Ionic Lys, Cys, Hcy, [244] Synthetic
Ionic probe Lys, AlCu
Cu2+,Cys,3+,Hcy,
Arg, 188 [244] Synthetic
IonicIonic
probeprobe Lys, Cys,Lys,Hcy, Cys, Hcy, 188 188[244] Synthetic
[244] Synthetic
GSH
Ionic probe Cu 2+,GSH
Lys, Cys,
Al3+, Hcy,
Arg,GSH 188 [244] Synthetic
GSH
Ionic probe GSHHcy,
Lys, Cys, 188 [244] Synthetic
Cu2+, Al3+, Arg,
GSH
Ionic probe
Ionic probe Lys, Cys,2+ Hcy,
Pd2+
2+ 188
189 [244]
[245] Synthetic
Synthetic
Ionic probeprobe
Ionic Pd Pd2+ 189 189[245] Synthetic
[245] Synthetic
Ionic probe Pd2+
GSH 189 [245] Synthetic
Ionic probe Pd2+ 189 [245] Synthetic
IonicIonic
Ionic probeprobe
probe Hg2+
Hg
2+
2+ Hg2+ 190,191 190,191
190,191 [246]
[246] Synthetic
[246]
Synthetic Synthetic
Ionic probe Hg2+ 190,191 [246] Synthetic
IonicIonic
Ionic probeprobe
probe Fe3+
Fe
3+
3+
Fe3+ 192
192 [247]
192[247] Synthetic
[247]
Synthetic Synthetic
Ionic probe Fe3+ 192 [247] Synthetic
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW Ionic probe Fe3+ 192 [247] 57 of 83
Synthetic
57 of 83

Ionic probe F- 193 [248] Synthetic
IonicIonic
probeprobe F-- F− 193 [248]
193[248] Synthetic Synthetic
Ionic probe F--, Ca2+ 194 [249] Synthetic

IonicIonic
probeprobe F- , Ca2+
2+ −
F , Ca2+ 194 194[249] Synthetic
[249] Synthetic
IonicIonic
probeprobe F--, CN-- F− , CN− 195 195[250] Synthetic
[250] Synthetic
Ionic probe F- , CN- 195 [250] Synthetic
Probe HSO3−, SO32- ion 196 [251] Synthetic

Probe HSO33−−, SO332-2- ion 196 [251] Synthetic
Probe HSO4− and SO42- CG, CA [256] Synthetic

Probe HSO44−− and SO442-2- CG, CA [256] Synthetic
Ionic probe F-, Ca2+ 194 [249] Synthetic
Int. J. Mol. Sci. 2020, 21, 4618 57 of 81

Ionic probe F-, CN- 195 [250] Synthetic
Ionic probe F-, CN- 195 [250] Synthetic
Ionic probeTable 2.F-,Cont.
CN- 195 [250] Synthetic
Molecular
Target
Probe HSO3−, SO32- ion 196 [251] Synthetic

Probe HSO3−, SO−32- ion 2− 196 [251] Synthetic
Probe
Probe HSO3−, HSO , SO3
SO32-3 ion 196 196 [251] Synthetic
[251] Synthetic
ion
Probe HSO4− and SO42- CG, CA [256] Synthetic

− 2-
Probe
Probe HSO4−HSO
and4SOand 4 CG, CG,
CA CA [256] [256]
Synthetic Synthetic
Probe HSO4− and SO442-2− CG, CA [256] Synthetic
umbelliferone,
R. Solanacearum, umbelliferone,
umbelliferone,
R. Solanacearum,
R. Solanacearum, esculetin,
umbelliferone,
Antibacterial genes fliA, flhC, esculetin,
Antibacterial R. genes fliA, flhC, esculetin, [261] Natural
Solanacearum, [261] Natural
Antibacterial genes fliA, flhC, daphnetin, [261] Natural
daphnetin,
esculetin,
biofilm formation
Antibacterial biofilm formation
genes fliA, flhC, xanthotol
daphnetin, [261] Natural
xanthotol
biofilm formation daphnetin,
biofilm formation xanthotol
xanthotol
O O
O O
O O O
H3CO O O O O
H3CO O O O O O
O O
H3CO O O O O O
H3CO O
OCH3O O O O
OCH3 OCH3
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW OCH3 58 of 83
O
O OCH
O 3 O
O Fungicide
OCH3
Colletotrichum
Fungicide Colletotrichum sp. sp. 197–202 [265]
197–202[265] Natural Natural
OCH
OCH O O O O
33 O
OCH3 OCH O O
O O O Fungicide
3
Botrytis cinerea,
Colletotrichum sp. 197–202 [265] Natural
Int. J. Mol.
O
Sci.
OCH2020, 21, x FOR
O O 3 OHPEER
O O REVIEW
O
Fungicide Colletotrichum sp. 197–202 58 of 83
[265] Natural
O OH OH
O O
OCH 3
OH
OH
Alternaria solani,
OCH 3 O
OCH 3 O
OH
OH Botrytis cinerea,
OCH
OH Gibberella zeae,
Int.
H COJ. Mol.
3 O Sci.
O 2020, 21,
O x FOR
3 O
O PEER REVIEW
O
Fungicide 203,204 [268] Synthetic58 of 83
3H CO OCH O O
3
O
OOCH O O
3 Alternaria solani,
H CO 3 O O
OCH
O O O
3 Rhizoctorzia solani,
3H CO O O O OCH
O O 3
Gibberella
Botrytiszeae,
cinerea,
OCH
Fungicide Cucumber anthrax
3
203,204 [268] Synthetic
Botrytis
Rhizoctorzia
Alternaria cinerea,
solani,
solani,
and Alternaria
Alternaria solani,
Cucumber Gibberella
anthrax
Gibberella zeae,
zeae,
Fungicide
FungicideAlternaria solani 203,204
203,204 [268][268]
SyntheticSynthetic
1 and Rhizoctorzia solani,
Alternaria
Rhizoctorzia
Cucumber solani,
anthrax
sorauer,
Cucumber
Alternaria Alternaria
andsolanianthrax
Fusarium
Fungicide: and Alternaria
sorauer,
oxysporum f.sp. solani205a–d
Alternaria [273] Synthetic
Fusarium
Alternaria solani
sorauer, Fusarium
Fungicide: vasinfectum
oxysporum f.sp.
Fungicide: oxysporum f.sp. and 205a–d205a–d[273] Synthetic
sorauer, [273] Synthetic
vasinfectum
and Fusarium
Fusarium
vasinfectum
Fusarium
Fungicide: moniliformemoniliforme
andoxysporum
Fusarium f.sp. 205a–d [273] Synthetic
moniliforme
vasinfectum
and Fusarium
moniliforme
MCF-7 human
MCF-7 human
Anticancer
Anticancer breast cancer207–213 207–213
[276] Synthetic
[276] Synthetic
breast cancer cells
cells
MCF-7 human
Anticancer 207–213 [276] Synthetic
breast cancer cells
MCF-7 human
breast cancer cells
Human lung
adenocarcinoma
Anticancer Human lung 214–224 [281] Synthetic
(A549/DDP) and
adenocarcinoma
Anticancer HeLa cells 214–224 [281] Synthetic
(A549/DDP)
Humanand
lung
HeLa cells
adenocarcinoma
(A549/DDP) and
HeLa cells
Catalyst 225 [282] Synthetic
moniliforme
MCF-7 human
breast cancer cells
Int. J. Mol. Sci. 2020, 21, 4618 MCF-7 human 58 of 81
breast cancer cells
Table 2. Cont.
Molecular
Structure Biological Activity Human lung Name/Number Ref. Origin
Target
adenocarcinoma
Anticancer Human lung 214–224 [281] Synthetic
(A549/DDP) and
Human lung
adenocarcinoma
adenocarcinoma
Anticancer
Anticancer HeLa cells 214–224 214–224
[281] Synthetic
[281] Synthetic
(A549/DDP)
(A549/DDP) and and
HeLa cells
HeLa cells
Catalyst
Catalyst 225 225 [282] [282]
Synthetic Synthetic
Catalyst 225 [282] Synthetic
4. Synthesis of Coumarin Scaffold

4. Synthesis of Coumarin
Knoevenagel ScaffoldWittig reaction, Claisen rearrangement, Heck reaction, Pechmann
condensation,
condensation and Perkin
Knoevenagel reaction Wittig
condensation, are widely used
reaction, methods
Claisen for the obtainment
rearrangement, Heck reaction,ofPechmann
Int.4.J. Synthesis
Mol. Sci. 2020, 21, x FOR Scaffold
of Coumarin PEER REVIEW 59 of 83
and have condensation
been and Perkin
widely reactionover
reviewed are widely
the yearsused [284,285].
methods for The the obtainment
increasingofinterest
coumarin in coumarins
derivatives
Knoevenagel and have been
condensation, widely
Wittig reviewed over
reaction, Claisenthe years [284,285]. The increasing interest in
as potential
procedures biologically
and active compounds led torearrangement,
the development Heck reaction, Pechmannprocedures and
of innovative
coumarins
condensation asapproaches
and potential aimed
Perkin reaction areatwidely
biologically the production
active compounds
used methods of
ledcoumarins
to the
for in high
the obtainment
development yield
of and in a sustainable
ofcoumarin
innovative
approaches
manner. In aimed
the next atparagraphs,
the production we of coumarins
will focus on in most
the high yield
recent and in a sustainable
advances in coumarin manner. In the
synthesis.
derivatives and have been widely reviewed over the years [284,285]. The increasing interest in
next paragraphs,
coumarins we will
as potential focus on active
biologically the most recent advances
compounds led to theindevelopment
coumarin synthesis.
of innovative
4.1. New Approaches in Coumarins Synthesis
4.1. New Approaches in Coumarins Synthesis
4.1.1.
4.1.1. Flow
Flow Chemistry
Chemistry and
and Immobilized
Immobilized Reagents
Reagents
In the past
In the past two
two decades,
decades, flow chemistry emerged
flow chemistry emerged has has aa promising
promising synthetic
synthetic technology,
technology, due to
due to
the many advantages it offers compared to the traditional batch method. In fact,
the many advantages it offers compared to the traditional batch method. In fact, by using continuousby using continuous
flow reactorsititis is
flow reactors possible
possible to control
to control reaction
reaction parameters,
parameters, such assuch as temperature,
temperature, stoichiometry,
stoichiometry, reaction
reaction time and others, very precisely, thus having the possibility to achieve
time and others, very precisely, thus having the possibility to achieve better and more reproducible better and more
reproducible reactions. Furthermore,
reactions. Furthermore, the great surfacethe great surface area/volume
area/volume ratio in flow
ratio in continuous continuous
reactorsflow reactors
often leads
often leads
to better to better
reaction reaction
yields. Thisyields. This new technology
new technology gives also the gives also the to
possibility possibility
work with to work with
hazardous
hazardous reagents and to perform superheated or pressurized reactions in
reagents and to perform superheated or pressurized reactions in safer conditions [286–288]. The safer conditions [286–
288]. The applications
applications of this technology
of this technology are numerous
are numerous and the improvement
and the improvement of chemicalofsynthesis
chemicalof synthesis
natural
of natural compounds
compounds is one of them.is oneIn of them.
2015, In 2015,
Li and Li and collaborators
collaborators developed a developed a two-stage
two-stage synthesis synthesis
of coumarins
of coumarins
via O-acetylationvia of
O-acetylation
salicylaldehyde of salicylaldehyde [289].occurred
[289]. The reaction The reaction occurred viaand
via O-acetylation O-acetylation and
intramolecular
intramolecular aldol-type followed
aldol-type condensation, condensation, followed byand
by dehydration dehydration and it was
it was performed performed
using two heatedusing two
coiled
heated coiled reactors (Scheme 1). Using this system, it was possible to reach
reactors (Scheme 1). Using this system, it was possible to reach a 120 g scale production. a 120 g scale production.
Scheme
Scheme 1.1. Set-up for the synthesis of coumarins via O-acetylation of salicylaldehyde in a continuous
flow reactor.
reactor.
The use of immobilized reagents in organic synthesis is gaining ever more attention, due to the
advantages offered by such type of reagents (for example, the simplification of product isolation and
purification). This synthetic strategy has seen some applications in the production of natural scaffolds
too. In 2018, Mhiri and co-workers proposed a synthetic pathway for coumarin derivatives involving
polymer supported reagents [290]. In the mentioned study, coumarin derivative A was prepared from
3-metoxy salicylaldheyde using reagents supported in a macroporous ion exchange resin, through the
hydrolysis of the two intermediates iminocoumarin B and unsaturated nitrile C (Scheme 2).
Scheme 1. Set-up for the synthesis of coumarins via O-acetylation of salicylaldehyde in a continuous
flow reactor.
The Sci.
Int. J. Mol. use2020,
of immobilized
21, 4618 reagents in organic synthesis is gaining ever more attention, due 59toofthe
81
advantages offered by such type of reagents (for example, the simplification of product isolation and
purification). This synthetic strategy has seen some applications in the production of natural scaffolds
too. In 2018,
too. In 2018, Mhiri
Mhiri and
and co-workers
co-workers proposed
proposed aa synthetic
synthetic pathway
pathway for
for coumarin
derivatives involving
involving
polymer supported reagents [290]. In the mentioned study, coumarin derivative
polymer supported reagents [290]. In the mentioned study, coumarin derivative A was A was prepared
prepared from
from
3-metoxy
3-metoxy salicylaldheyde using reagents
salicylaldheyde using reagents supported in aa macroporous
supported in macroporous ion
ion exchange
exchange resin,
resin, through
through the
the
hydrolysis
hydrolysis of the two intermediates iminocoumarin B and unsaturated nitrile C (Scheme 2).2).
of the two intermediates iminocoumarin B and unsaturated nitrile C (Scheme
Int. J. Mol. Sci. 2020, 21, x FOR PEER2.REVIEW

Scheme Coumarin synthesis with polymer-supported reagent. 60 of 83
The first
first step
stepprovided
provided 2.the
Schemethe preparation
preparation
Coumarin of of polymer
polymer
synthesis supported
supported phenate
phenate
with polymer-supported ionsions
that that
reagent. then then reacted
reacted with
with phenylacetonitrile,
phenylacetonitrile, leading
leading to thetoformation
the formation of iminocoumarin
of iminocoumarin and unsaturated
and unsaturated nitrile,nitrile,
whichwhich
were
were
finallyfinally deblocked
deblocked from from the resin
the resin with with chloroform.
chloroform. TheThe
acidacid hydrolysis
hydrolysis of the
of the obtained
obtained mixture
mixture of
of compounds B and C gave coumarin derivative A in very good
compounds B and C gave coumarin derivative A in very good yield (95%). yield (95%).
4.1.2. Photocatalysis
It is worth
worth speaking
speaking about
about another
another emerging
emerging strategy
strategy inin organic
organicsynthesis:
synthesis: photocatalysis.
photocatalysis. In
Metternich and
2015, Metternich andGilmour
Gilmourutilized
utilizedthis
this approach
approach to to emulate
emulate coumarins
coumarins biosynthesis,
biosynthesis, using
using (−)-
(−)-riboflavin
riboflavin as aas a photocatalyst
photocatalyst [291].
[291]. In work,
In this this work, two discrete
two discrete activation
activation modes modes of (−)-riboflavin
of (−)-riboflavin were
were sequentially
sequentially exploited
exploited to induce
to induce the the isomerization
isomerization andand cyclisation
cyclisation E-cinnamicacids
ofofE-cinnamic acids used
used as
starting material (Scheme 3), thus overcoming the use of phenol-derived and pre-functionalized aryl
rings as starting materials.
Scheme 3. (a) Photocatalyzed synthesis of coumarins; (b) Chemical structure of (−) Riboflavin.
Similarly, in 2019, Song and co-workers reported a one-pot photoredox-catalyzed protocol

Scheme
to achieve 3. (a) Photocatalyzed
a series synthesiscoumarins
of 3-fluoroalkylated of coumarins; (b) Chemical
starting from structure of (-) Riboflavin. esters
ortho-hydroxycinnamic
(Scheme 4) [292]. ortho-Hydroxycinnamic esters and BrCF2 COR’ (or other commercially available
Similarly, in 2019,
perfluoroalkylated Songresources)
radical and co-workers reportedtoa 30W
were exposed one-pot photoredox-catalyzed
blue LEDs irradiation under protocol to
argon
achieve a series
atmosphere ofh.;
for 12 3-fluoroalkylated
fac-Ir(ppy)3 was coumarins
found to bestarting
the bestfrom
amongortho-hydroxycinnamic esters (Scheme
the tested catalysts, whereas CH3 CN,
4) [292]. ortho-Hydroxycinnamic esters and BrCF 2COR’ (or other commercially available
DMF, DMSO and THF were all suitable solvents for this reaction. K2 CO3 , Et3 N and DIPEA were all
perfluoroalkylated
able to neutralize HBr radical resources)
produced during were exposed
the process. toprocedure
This 30W bluegaveLEDs irradiation under
3-fluoroalkylated argon
coumarins
atmosphere
in good yieldsforon12milligram
h.; fac-Ir(ppy) 3 was found to be the best among the tested catalysts, whereas
and gram scales.
CH3CN, DMF, DMSO and THF were all suitable solvents for this reaction. K2CO3, Et3N and DIPEA
were all able to neutralize HBr produced during the process. This procedure gave 3-fluoroalkylated
coumarins in good yields on milligram and gram scales.
perfluoroalkylated radical resources) were exposed to 30W blue LEDs irradiation under argon
atmosphere for 12 h.; fac-Ir(ppy)3 was found to be the best among the tested catalysts, whereas
CH3CN, DMF, DMSO and THF were all suitable solvents for this reaction. K2CO3, Et3N and DIPEA
were all able to neutralize HBr produced during the process. This procedure gave 3-fluoroalkylated
coumarins in good yields on milligram and gram scales.
Int. J. Mol. Sci. 2020, 21, 4618 60 of 81
Scheme 4. Photoredox-catalyzed synthesis of 3-fluoroalkylated coumarins.
Another example Scheme is 4.the bichromatic synthesis

Photoredox-catalyzed of coumarins
synthesis proposed coumarins.
of 3-fluoroalkylated by Eivgi and collaborators
in 2017 [293]. In this work, 2-nitrobenzyl-protected 2-hydroxystyrenes and acrylates underwent a
UV-AAnother
(380 nm) photoinduced
example cross metathesis
is the bichromatic synthesis(CM) reaction,proposed
of coumarins in the presence
by Eivgiofand ruthenium as a
collaborators
catalyst. In this
in 2017 [293]. stepwork,
In this a 1-pyrenecarboxaldheyde
2-nitrobenzyl-protected solution was used as and
2-hydroxystyrenes a UV filter, without
acrylates whom
underwent the
a UV-
catalyst
A (380 nm) would be permanently
photoinduced crossinactivated
metathesisdue(CM) toreaction,
phenol deprotection andof
in the presence phenolate
ruthenium chelation to the
as a catalyst.
ruthenium.
In this step After CM, the UV filter was removed
a 1-pyrenecarboxaldheyde solution wasand irradiation
used as a UVwith UV-C
filter, light (254
without nm) the
whom led to more
catalyst
complex
would bestructures,
permanently such inactivated
as coumarins.dueIn to
fact, irradiation
phenol with UV-C
deprotection andlight started achelation
phenolate three-reactions
to the
chain—photodeprotection
ruthenium. After CM, the of UVthe intermediate,
filter was removedE/Z isomerization
and irradiation and cyclization
with UV-C light to form
(254 the
nm)desired
led to
coumarins
Int. J. Mol.
more complex (Scheme
Sci. 2020, 21, 5).
x FOR
structures, PEER REVIEW
such as coumarins. In fact, irradiation with UV-C light started a three- 61 of 83
reactions chain—photodeprotection of the intermediate, E/Z isomerization and cyclization to form
the desired coumarins (Scheme 5).
Scheme 5. (a) Bichromatic synthesis of coumarins; (b) chemical structure of catalyst A.
4.1.3. Solvent-Free Reactions

Scheme 5. (a) Bichromatic synthesis of coumarins; (b) chemical structure of catalyst A.
Chemists usually carry out reactions in solution, following the Aristotelian principle “Corpora non
agunt
4.1.3.nisi fluida seu soluta”
Solvent-Free or in other and more contemporary words “Compounds do not react unless
Reactions
fluid or if dissolved” [294]. However, this is not always completely accurate, because many solventless
Chemists usually carry out reactions in solution, following the Aristotelian principle “Corpora
reactions proceed efficiently. An enhancement in kinetics, owed to the different concentrations of
non agunt nisi fluida seu soluta” or in other and more contemporary words “Compounds do not react
reactants given the lack of solvents, bring actually to a higher reactivity and, where necessary, milder
unless fluid or if dissolved” [294]. However, this is not always completely accurate, because many
experimental conditions. At the same time, the absence of solvents provides the chance to heat over the
solventless reactions proceed efficiently. An enhancement in kinetics, owed to the different
boiling point of the most common solvents and to exploit the benefits of microwave assisted irradiation.
concentrations of reactants given the lack of solvents, bring actually to a higher reactivity and, where
The occurrence of efficient solid-state reactions shows that the reacting molecules are able to move
necessary, milder experimental conditions. At the same time, the absence of solvents provides the
freely in the solid state, whereas monitoring of the reaction is possible thanks IR and UV spectra in the
chance to heat over the boiling point of the most common solvents and to exploit the benefits of
solid state [295,296]. The most remarkable advantage of solvent-free reaction, in all probability, is the
microwave assisted irradiation. The occurrence of efficient solid-state reactions shows that the
noteworthy cut in the waste production associated with solvents handling, resulting in a more effective
reacting molecules are able to move freely in the solid state, whereas monitoring of the reaction is
and ecological chemical process. There are three solvent-free techniques: (1) reactions conducted
possible thanks IR and UV spectra in the solid state [295,296]. The most remarkable advantage of
on mineral supports, (2) reaction without any solvent, support or catalyst and (3) solid-liquid phase
solvent-free reaction, in all probability, is the noteworthy cut in the waste production associated with
transfer catalysis [297].
solvents handling, resulting in a more effective and ecological chemical process. There are three
The experimental conditions required for the classical synthesis of coumarin derivatives are, in
solvent-free techniques: (1) reactions conducted on mineral supports, (2) reaction without any
some cases, drastic, as in the production of hymecromone via Pechmann condensation where the final
solvent, support or catalyst and (3) solid-liquid phase transfer catalysis [297].
compound is obtain by stirring a mixture of resorcinol and ethyl acetoacetate in concentrated H2SO4
for 12–24 h [295]. Solvent-free reactions seemed to be a more ecological option. Consequently, in 2001,
some cases, drastic, as in the production of hymecromone via Pechmann condensation where the
Sugino and Tanaka proposed the synthesis of several coumarin derivatives both via Pechmann and
final compound is obtain by stirring a mixture of resorcinol and ethyl acetoacetate in concentrated
H2SO4 for 12–24 h [295]. Solvent-free reactions seemed to be a more ecological option. Consequently,
in 2001, Sugino and Tanaka proposed the synthesis of several coumarin derivatives both via
Pechmann and Knoevenagel condensation reactions [298]. For example, p-toluensolfonic acid was
employed as a catalyst and the reaction mixture was heated at 60 °C for 10 min. without solvent,
some cases, drastic, as in the production of hymecromone via Pechmann condensation where the
final compound is obtain by stirring a mixture of resorcinol and ethyl acetoacetate in concentrated
H2SO4 for 12–24 h [295]. Solvent-free reactions seemed to be a more ecological option. Consequently,
in 2001, Sugino and Tanaka proposed the synthesis of several coumarin derivatives both via
Int. J. Mol. Sci. 2020, 21, 4618 61 of 81
Pechmann and Knoevenagel condensation reactions [298]. For example, p-toluensolfonic acid was
employed as a catalyst and the reaction mixture was heated at 60 °C for 10 min. without solvent,
giving after the
Knoevenagel work-up 7-hydroxy-4-methylcoumarin
condensation in 98%
reactions [298]. For example, yield (Schemeacid
p-toluensolfonic 6). was employed as a
catalyst and the reaction mixture was heated at 60 ◦ C for 10 min. without solvent, giving after the
work-up 7-hydroxy-4-methylcoumarin in 98% yield (Scheme 6).
Knoevenagel condensation was also studied under solventless conditions and efficiently
brought to the production of both coumarins and benzocoumarin derivatives [298].
Another
Int. J. Mol. methodology
Sci. 2020, was
21, x FOR PEER proposed in 2012 by Kalita and Kumar, that exploited the Pechmann
REVIEW 62 of 83
reaction using trifluoromethanesulfonic acid functionalized Zr-TMS (Zr-TMS, zirconia-based
Scheme 6. Solvent-free synthesis of 7-hydroxy-4-methylcoumarin.
transition metal oxide
Knoevenagel mesoporous
condensation wasmolecular sieves)under
also studied catalysts, under solvent-free
solventless heterogeneous
conditions and efficiently
Scheme 6. Solvent-free synthesis of 7-hydroxy-4-methylcoumarin.
catalytic
brought Knoevenagel condensation was also studied under solventless conditions and efficiently [296].
conditions.
to the The
production product
of both selectivity
coumarins was
andalways found
benzocoumarin to be about 100%
derivatives (Scheme
[298]. 7) brought
to theAnother
production methodology was proposed
of both coumarins in 2012 by Kalita
and benzocoumarin and Kumar,
derivatives that exploited the Pechmann
[298].
reaction using
Another trifluoromethanesulfonic
methodology was proposed inacid 2012 functionalized Zr-TMS
by Kalita and Kumar, that(Zr-TMS,
exploited zirconia-based
the Pechmann
transition
reaction metal
using oxide mesoporous molecular
trifluoromethanesulfonic sieves) catalysts,
acid functionalized Zr-TMSunder solvent-free
(Zr-TMS, heterogeneous
zirconia-based transition
catalytic
metal conditions.
oxide mesoporousThe product selectivity
molecular sieves)was always under
catalysts, found solvent-free
to be about 100% (Scheme 7) catalytic
heterogeneous [296].
conditions. The product selectivity was always found to be about 100% (Scheme 7) [296].
Scheme 7. Solvent-free Pechmann condensation with Zr-TMS-TFA as catalyst.
More recently,
Scheme a novel series of
7. Solvent-free hetero-annulated
Pechmann coumarins
condensation was obtained
with Zr-TMS-TFA by Ebrahimi and
as catalyst.
colleagues through a one-pot reaction under solvent-free conditions and evaluated as AChE/BuChE
More As
inhibitors. recently,
acidic
Scheme
acatalyst
novel series of
nano-silica
7. Solvent-free
hetero-annulated
sulfuric
Pechmann acid wascoumarins
condensation employed was obtained
and the
with Zr-TMS-TFA
by Ebrahimi and
versatility
as catalyst. of the system
colleagues through a one-pot reaction under solvent-free conditions and
was assayed on appropriated benzaldehydes, dimedone and 4-hydroxycoumarin (Scheme evaluated as AChE/BuChE
8) [299].
inhibitors.
Also More As
noteworthyacidic catalyst
recently,isa the
novel nano-silica
application, sulfuric acid was employed
once again, of acoumarins
series of hetero-annulated and
reusable was the versatility
heterogeneous of the system
obtained bycatalyst,
Ebrahimi which
and
was assayedthrough
contributed
colleagues onthe
to appropriated
general benzaldehydes,
environmental
a one-pot dimedone
sustainability
reaction under and 4-hydroxycoumarin
of conditions
solvent-free the processand
thanks to(Scheme 8) [299].
the possible
evaluated Also
recovery
as AChE/BuChE
noteworthy
and is acidic
reuse [300].
inhibitors. As the application, once again,
catalyst nano-silica of a reusable
sulfuric acid washeterogeneous
employed and catalyst, which contributed
the versatility of the systemto
the
wasgeneral
assayed environmental
on appropriated sustainability of the process
benzaldehydes, thanks
dimedone andto4-hydroxycoumarin
the possible recovery and reuse
(Scheme [300].
8) [299].
Also noteworthy is the application, once again, of a reusable heterogeneous catalyst, which
contributed to the general environmental sustainability of the process thanks to the possible recovery
OH
and reuse [300]. O O nano-SSA
ArCHO O O
Solvent-free
Ar
O O 100°C
2-3h O O
OH
O O nano-SSA 78-60%
O yields O
ArCHO
Solvent-free
Scheme 8. Solvent-free synthesis Ar
O O of hetero-annulated
100°C coumarins.
2-3h O O
Scheme
Another solventless 8. Solvent-free
procedure synthesis
worth noting of one
is the hetero-annulated
presented bycoumarins.
Kour and Paul, which exploited
Lewis acid grafted sulfonated carbon@titania composite as catalyst for the 78-60% yields
Pechmann condensation.
This particular kind of catalyst and its efficacy had been already studied by the teamand
Another solventless procedure worth noting is the one presented by Kour Paul,
for the which
synthesis
exploited Lewis acid grafted sulfonated carbon@titania composite as catalyst
of 4H-pyrimido[2,1-b]benzothiazoles and benzoxanthenones under solvent-free conditions, so the for the Pechmann
condensation. This particular
Scheme 8.kind of catalyst
Solvent-free and its
synthesis efficacy had been
of hetero-annulated already studied by the team
coumarins.
for the synthesis of 4H-pyrimido[2,1-b]benzothiazoles and benzoxanthenones under solvent-free
conditions,
Anotherso solventless
the one-potprocedure
synthesis worth
of coumarin
notingderivatives
is the one was the natural
presented nextand
by Kour stepPaul,
(Scheme 9)
which
[301,302].
exploited Because the grafted
Lewis acid catalyst sulfonated
reusability contributes
carbon@titaniato reduce the cost
composite of the practical
as catalyst for the application
Pechmann
processes, the reusability
condensation. of thekind
This particular catalyst was investigated
of catalyst in the had
and its efficacy Pechmann condensation.
been already studied The catalyst,
by the team
Int. J. Mol. Sci. 2020, 21, 4618 62 of 81
one-pot synthesis of coumarin derivatives was the natural next step (Scheme 9) [301,302]. Because
the
63 of 83
catalyst reusability contributes to reduce the cost of the practical application processes, the reusability
of the catalyst
exhibited almostwas theinvestigated in the
same TG curve as Pechmann condensation.
the fresh one, depicting thatThe
thecatalyst,
presentrecovered by filtration,
heterogeneous system
after five catalytic runs had a mild decrease in the catalytic
showed high thermal stability after successive reaction cycles [302]. activity and exhibited almost the same TG
curve as the fresh one, depicting that the present heterogeneous system showed high thermal stability
after successive reaction cycles [302].
Scheme 9. Lewis acid grafted sulfonated carbon@titania composite as catalyst for the
Pechmann condensation.
Scheme 9. Lewis acid grafted sulfonated carbon@titania composite as catalyst for the Pechmann
In order to obtain a different and ecological procedure for the synthesis of 3-substituted coumarins
condensation.condensation, Ghomi and Akbarzadeh developed a procedure starting from various
via Knoevenagel
salicylaldehydes
Moreover, given and the 1,3-dicarbonyl
mild experimental compounds.conditions,Theythere used MgFe
were 2no O4 by-products
nanoparticlesattributable
as an efficient to
In order to obtain a different and ecological procedure for the synthesis of 3-substituted
catalyst under solvent-free
decomposition or polymerization.condition and ultrasound irradiation (Scheme 10).
coumarins via Knoevenagel condensation, Ghomi and Akbarzadeh developed a procedure starting
from various salicylaldehydes and 1,3-dicarbonyl compounds. They used MgFe2O4 nanoparticles as
an efficient catalyst under solvent-free condition and ultrasound irradiation (Scheme 10).
Basically, ultrasound irradiation increased the dimension of the single bubbles together with the
number of active cavitation bubbles. The resulting effect was expected to be a higher maximum
collapse temperature and a faster synthesis of coumarin compounds by Knoevenagel reaction.
Comparing ultrasound irradiation to conventional heating the research team demonstrated that the
first method allowed to obtain higher yields in shorter times, probably because of the shock wave
and Scheme Ionic liquids
microjet10.generated byasthe
an acidic catalystFurthermore,
cavitation. in the regioselective
with synthesis
this method,of pyrano[3,2-c]
MgFe2O4coumarins
nanoparticles
Scheme
under 10. Ionic
solvent-free liquids
conditions. as an acidic catalyst in the regioselective
were dispersed in the reaction and provided more sites for the construction of cavity over their synthesis of pyrano[3,2-c]
coumarins
surface. under solvent-free
The catalyst was recovered conditions.
via magnetic separation and reused up to 6 cycles without
Basically, ultrasound irradiation increased the dimension of the single bubbles together with the
relevant loss of activity [303].
number of active cavitation
Noroozizadeh bubbles. The
and co-workers resulting
focused their effect
efforts wasonexpected
the synthesisto be aofhigher maximumthanks
bis-coumarins collapseto
The convenience given by the removal of the catalyst by means of an external magnetic field is
temperature and a faster synthesis of coumarin compounds
a different catalyst, acetic acid functionalized poly(4-vinylpyridinum) bromide (APVPB), in by Knoevenagel reaction. Comparing
a significant improvement in the work-up of a reaction mixture. Consequently, another magnetic
ultrasound of
continuous irradiation
a previous towork
conventional heatingteam
of the research the research team demonstrated
on the preparation of Brønsted that the ionic
acidic first method
liquids
nano structured catalyst was employed for the synthesis of coumarin nucleus, this time through
allowed
and solidtosalts
obtain higher The
[312,313]. yieldsaimin was
shorter the times, probably
development ofbecause
a novel of andtheefficient
shock wave and microjet
procedure for the
Pechmann condensation. In 2019, Pakdel and co-workers proposed the synthesis of coumarin
generatedofbybis-coumarins
synthesis the cavitation.which Furthermore,
lacked the with this method,
classical drawbacks MgFe2O4
as the usenanoparticles
of solvents,were highdispersed
costs and
derivatives by leveraging Fe3O4@Boehmite-NH2-CoII NPs as a catalyst, under solvent-free conditions
in the
low reaction
yields [314]. and provided more sites for the construction of cavity over their surface. The catalyst
that resulted the best choice comparing the results obtained with classic solvents. The catalyst could
was recovered via magnetic separation and reused up to 6 cycles without relevant loss of activity [303].
be recovered easily and reused up to six times. However, no desired coumarin derivatives were
4.1.4.The
Microwave
convenience Assisted
givenReactions
by the removal of the catalyst by means of an external magnetic field is a
observed in the case of phenols bearing electron-withdrawing substituents (such as -Cl and –NO2),
significant improvement in the work-up of a reaction mixture. Consequently, another magnetic nano
Temperature
behavior attributedistoa the keyproposed
parameter in almostofthe
mechanism thetotality
reactionof[304].
chemical reactions involving both
structured catalyst was employed for the synthesis of coumarin nucleus, this time through Pechmann
inorganic
In theand
fieldorganic compounds.
of solvent-free reactions,Generally, most organic
the application of ionicreactions
liquids as arecatalyst
performed for the heating
synthesis by
condensation. In 2019, Pakdel and co-workers proposed the synthesis of coumarin derivatives by
traditional
of coumarin heat transferdeserves
scaffolds equipment like oil baths,
a stand-alone status.sand baths
Ionic and heating
liquids (ILs) have jackets.
become Theincreasingly
drawbacks
leveraging Fe3 O4 @Boehmite-NH2 -CoII NPs as a catalyst, under solvent-free conditions that resulted
associated
popular inwith these
the last heating
decades astechniques are the slowness,
a safer alternative to classic the possible
solvent development
systems, of temperature
in a perspective of more
the best choice comparing the results obtained with classic solvents. The catalyst could be recovered
gradient
sustainable within the sample
chemical and consequent
processes. Most ILs possess local overheating
a number ofthat could lead
properties that to the formation
made of by-
them appealing,
easily and reused up to six times. However, no desired coumarin derivatives were observed in the case
products. Microwave technology started to emerge in inorganic
proving to be non-flammable, to possess a negligible vapor-pressure, that means that solvent chemistry since the late 1970s and
of phenols bearing electron-withdrawing substituents (such as -Cl and –NO2), behavior attributed to
almost a decade
evaporation after attracted
is eliminated and to thedissolve
attention of organic
a wide range chemists
of organic [315].
and Compared to the traditional
inorganic compounds [305].
the proposed mechanism of the reaction [304].
heating,
Furthermore,microwave dielectric
since ionic liquidsheating has severalsalts,
are low-melting advantages:
they are(1) higher
made heating
at least from rates,
two (2) no direct
components
In the
contact field ofthe
between solvent-free
source ofreactions,
energy the application
and thethus
reacting of chemicals,
ionic liquids(3) as catalyst for the synthesis of
which can be varied (the anion and the cation), the solvents could bepossible
designedselective heating,
with a particular
coumarin
because scaffolds deserves a stand-alone status. Ionic liquids (ILs) have become increasingly popular
end use reacting
in mindchemicalsor possessinginteract differentlyset
a particular with of the commonly
properties used
[306]. microwave
However, theirradiations
use in largeand
in the
(4) last decades
rapid increase asofa safer
the alternative to also
temperature classic solvent systems, in a in
perspective of more sustainable
quantities is restricted by some limitations likeabove boiling
biodegradability, point toxicitypressurized
and high costs systems [316].
[307–309].
chemical processes.
Microwave Mostoften
ILs possess a number of properties that made as them appealing, proving
Mahato andirradiation
colleagues is in 2017 usedassociated with other
ionic liquids as antechnologies,
acidic catalystsuch ionic liquids synthesis
in the regioselective and neat
to be non-flammable,
reactions (already to
mentioned possess a negligible
in thesolvent-free vapor-pressure,
previous paragraph) that
[295,317]. means that
In thegiven solvent
late 1990s evaporation
progresses in theis
of pyrano[3,2-c] coumarins under conditions. In particular, the fact that Brønsted
eliminated
classic and
coumarins to dissolve a wide range of organic and inorganic compounds [305]. Furthermore,
acidic ion liquids synthesis via microwave
(BAILs) acquired recognitionirradiation
in the werefield reported
of catalysis, by Singh and co-workers,
the research team reportedwho
confirmed
the catalytic a reduction in reaction
effect of 1-butane time of
sulfonic the Pechmann condensation
acid-3-methylimidazolium and by
tosylate, Saidi and colleagues,
[BSMIM]OTs (BAIL-1),
who
on the demonstrated
tandem reaction the between
improvement in the synthesis
4-hydroxycoumarin and of α,β-unsaturated
pyranocoumarins and furanocoumarins
carbonyl compounds for
through Claisen
the formation rearrangement [317,318].(Scheme
of pyrano[3,2-c]coumarins Ten years 10)later, Valizadeh
[310,311]. and Shockravi
In addition to the highpushed further
yields and the
the opportunitiesthis
regioselectivity, given by microwave
procedure allowed heating
to avoid combining
column it with a task-specific
chromatography imidazolium-based
for further purification.
phosphinite ionic liquid (IL-OPPh2) for the synthesis of E-cinnamates and coumarin derivatives via
Moreover,
Int. given
J. Mol. Sci. 2020, the
mild experimental conditions, there were no by-products attributable
21, 4618 63 of 81to
decomposition or polymerization.
since ionic liquids are low-melting salts, they are made at least from two components which can
be varied (the anion and the cation), thus the solvents could be designed with a particular end use
in mind or possessing a particular set of properties [306]. However, their use in large quantities
is restricted by some limitations like biodegradability, toxicity and high costs [307–309]. Mahato
and colleagues in 2017 used ionic liquids as an acidic catalyst in the regioselective synthesis of
pyrano[3,2-c] coumarins under solvent-free conditions. In particular, given the fact that Brønsted acidic
ion liquids (BAILs) acquired recognition in the field of catalysis, the research team reported the catalytic
effect of 1-butane sulfonic acid-3-methylimidazolium tosylate, [BSMIM]OTs (BAIL-1), on the tandem
Scheme 10. Ionic liquids as an acidic catalyst in the regioselective synthesis of pyrano[3,2-c]
reaction between 4-hydroxycoumarin and α,β-unsaturated carbonyl compounds for the formation of
coumarins under solvent-free conditions.
pyrano[3,2-c]coumarins (Scheme 10) [310,311]. In addition to the high yields and the regioselectivity, this
procedure allowed to avoid column chromatography for further purification. Moreover, given the mild
Noroozizadeh and co-workers focused their efforts on the synthesis of bis-coumarins thanks to
experimental conditions, there were no by-products attributable to decomposition or polymerization.
a different catalyst, acetic acid functionalized poly(4-vinylpyridinum) bromide (APVPB), in
Noroozizadeh and co-workers focused their efforts on the synthesis of bis-coumarins thanks to a
continuous of a previous work of the research team on the preparation of Brønsted acidic ionic liquids
different catalyst, acetic acid functionalized poly(4-vinylpyridinum) bromide (APVPB), in continuous
and solid salts [312,313]. The aim was the development of a novel and efficient procedure for the
of a previous work of the research team on the preparation of Brønsted acidic ionic liquids and solid
synthesis of bis-coumarins which lacked the classical drawbacks as the use of solvents, high costs and
salts [312,313]. The aim was the development of a novel and efficient procedure for the synthesis
low yields [314].
of bis-coumarins which lacked the classical drawbacks as the use of solvents, high costs and low
yields [314].
4.1.4. Microwave Assisted Reactions
4.1.4. Microwave
Temperature Assisted
is a key Reactions
parameter in almost the totality of chemical reactions involving both
inorganic
Temperature is a key parameter Generally,
and organic compounds. in almost the mosttotality
organic of reactions
chemical are performed
reactions heating
involving bothby
traditional heat transfer equipment like oil baths, sand baths and heating
inorganic and organic compounds. Generally, most organic reactions are performed heating by jackets. The drawbacks
associatedheat
traditional withtransfer
these heating techniques
equipment like oilare the slowness,
baths, sand baths theand
possible
heatingdevelopment
jackets. The of drawbacks
temperature
gradient within the sample and consequent local overheating that could lead
associated with these heating techniques are the slowness, the possible development of temperature to the formation of by-
gradient within the sample and consequent local overheating that could lead to the formation and
products. Microwave technology started to emerge in inorganic chemistry since the late 1970s of
almost a decade
by-products. after attracted
Microwave the attention
technology started to ofemerge
organicinchemists
inorganic[315]. Compared
chemistry since tothe
thelate
traditional
1970s
heating,
and almostmicrowave
a decadedielectric heatingthe
after attracted hasattention
several advantages:
of organic (1) higher [315].
chemists heatingCompared
rates, (2) notodirect
the
contact between the source of energy and the reacting chemicals, (3) possible
traditional heating, microwave dielectric heating has several advantages: (1) higher heating rates, selective heating,
because
(2) reacting
no direct contact chemicals
betweeninteract
the source differently
of energy with
andthe
thecommonly used microwave
reacting chemicals, radiations
(3) possible and
selective
(4) rapid increase of the temperature also above boiling point in pressurized
heating, because reacting chemicals interact differently with the commonly used microwave radiations systems [316].
Microwave
and (4) rapidirradiation
increase ofisthe often associated also
temperature withabove
other boiling
technologies,
point insuch as ionic liquids
pressurized systems and neat
[316].
reactions (already mentioned in the previous paragraph) [295,317]. In the
Microwave irradiation is often associated with other technologies, such as ionic liquids and neat late 1990s progresses in the
classic coumarins synthesis via microwave irradiation were reported by Singh
reactions (already mentioned in the previous paragraph) [295,317]. In the late 1990s progresses in the and co-workers, who
confirmed
classic a reduction
coumarins synthesis in reaction time of irradiation
via microwave the Pechmann werecondensation and byand
reported by Singh Saidi and colleagues,
co-workers, who
who demonstrated the improvement in the synthesis of pyranocoumarins
confirmed a reduction in reaction time of the Pechmann condensation and by Saidi and colleagues, and furanocoumarins
through
who Claisen rearrangement
demonstrated the improvement [317,318].
in theTen years later,
synthesis Valizadeh and Shockravi
of pyranocoumarins pushed further
and furanocoumarins
the opportunities
through given by microwave
Claisen rearrangement [317,318]. heating combining
Ten years it with a task-specific
later, Valizadeh and Shockravi imidazolium-based
pushed further
phosphinite ionic liquid (IL-OPPh 2) for the synthesis of E-cinnamates and coumarin derivatives via
the opportunities given by microwave heating combining it with a task-specific imidazolium-based
the one-pot Horner–Wadsworth–Emmons–type reaction (Scheme 11).
phosphinite ionic liquid (IL-OPPh2 ) for the synthesis of E-cinnamates and coumarin derivatives via
the one-pot Horner–Wadsworth–Emmons–type reaction (Scheme 11).
CHO IL-OPPh2, NaOMe COOMe

+
+ Cl COOMe
R OH MW (400 W, 110 °C) R O O R OH
79-83% yields
Scheme11.11.Imidazolium-based
Scheme Imidazolium-based phosphinite
phosphinite ionic
ionicliquid
liquid(IL-OPPh 2 )2)for
(IL-OPPh forthe
themicrowave
microwaveassisted
assisted
synthesis
synthesisofofcoumarins.
coumarins.
The
Thesame reactions
same werewere
reactions in parallel conducted
in parallel with traditional
conducted heating and
with traditional microwave
heating and irradiation:
microwave
the former technique showed considerably longer reaction times (13–16 h vs. 10–12 min) and
irradiation: the former technique showed considerably longer reaction times (13–16 h vs 10–12 lower
min)
yields (65–60% vs. 79–83%). Furthermore, IL-OPPh2 worked at the same time as reaction medium and
Int. J. Mol. Sci. 2020, 21, 4618 64 of 81

and lower yields (65–60% vs 79–83%). Furthermore, IL-OPPh2 worked at the same time as reaction
medium and reagent as the research team already demonstrated in a previous study focused on the
synthesis
reagent asofthecoumarin
researchnucleus via Knoevenagel
team already demonstrated and inWitting reactions
a previous study in focused
ionic liquids
on the [319,320].
synthesis of
In 2016,
coumarin Fiorito
nucleus viaand colleagues merged
Knoevenagel and Wittingthe advantages
reactions inof solvent-free
ionic reactions with microwave
liquids [319,320].
heating for the
In 2016, synthesis
Fiorito of severalmerged
and colleagues coumarin thederivatives.
advantages of They started from
solvent-free variously
reactions with substituted
microwave
phenols for
heating andthe propiolic
synthesis acids in the presence
of several coumarinofderivatives.
ytterbium triflate hydratefrom
They started 10% variously
mol as catalyst [321].
substituted
During the
phenols andlast thirty years,
propiolic acidstriflates of metalsofbelonging
in the presence ytterbiumtotriflate
the lanthanide
hydrate series
10% mol have asbeen regarded
catalyst [321].
as effective
During the lastwater
thirtytolerant
years,recyclable
triflates of Lewis
metalsacids. Thesetometals
belonging have been
the lanthanide discovered
series have been toregarded
catalyze
different
as effectivecarbon–carbon
water tolerant and carbon–heteroatom
recyclable Lewis acids.bond These formation
metals have reactions, providing the
been discovered desired
to catalyze
products in excellent yields by means of a green chemical approach
different carbon–carbon and carbon–heteroatom bond formation reactions, providing the desired [322,323]. Furthermore, Wang
and colleagues
products already
in excellent employed
yields by means this particular
of a greencatalyst
chemical forapproach
the synthesis of 4-methylcoumarins
[322,323]. Furthermore, Wang via
Pechmann
and colleaguescondensation
already employed[324]. Fiorito and co-workers
this particular catalystdeveloped for the of
for the synthesis first time a synthetic
4-methylcoumarins
procedure
via Pechmann to obtain different substituted
condensation [324]. Fiorito coumarins in 2 min.developed
and co-workers under microwave irradiation
for the first time a in 91-98%
synthetic
yields [321].
procedure toMoreover,
obtain differentthe research teamcoumarins
substituted recovered in the2 ytterbium
min. undertriflate catalyst
microwave and demonstrated
irradiation in 91-98%
that no[321].
yields loss of activity occurred
Moreover, the research during
team several cycles.
recovered the ytterbium triflate catalyst and demonstrated
that noIn the
losssame year, Bouasla
of activity occurredetduringal. compared
several the efficacy of different heterogeneous acidic catalysts
cycles.
in theInPechmann
the same year, synthesis
Bouaslaof 4-methylcoumarin
et al. compared the and 7-hydroxy-4-methylcoumarin
efficacy of different heterogeneous when coupled
acidic by
catalysts
microwave heating. The efficacy of different Amberylst-type catalyst
in the Pechmann synthesis of 4-methylcoumarin and 7-hydroxy-4-methylcoumarin when coupled in the Pechmann synthesis of 7-
hydroxy-4-methylcoumarin
by microwave heating. The efficacy was already pointed
of different out by Sabou
Amberylst-type and in
catalyst colleagues
the Pechmann in 2005 [325].
synthesis
Therefore,
of the catalytic performance
7-hydroxy-4-methylcoumarin wasofalready
Amberlyst-15
pointedwas out compared
by Sabou with zeolite H-βinand
and colleagues 2005sulfonic
[325].
acid functionalized
Therefore, the catalytichybrid
performancematerial TS-OS-SO3H,was
of Amberlyst-15 performing
compared with the reaction
zeolite H-β under solvent-free
and sulfonic acid
conditions, at 130
functionalized °C for
hybrid 20 minTS-OS-SO
material in a microwave
3 H, reaction
performing tube.
the Amberlyst-15
reaction under showed a
solvent-free higher catalytic
conditions, at
◦
activity (97% yield against 21–44% obtained with two others catalysts)
130 C for 20 min in a microwave reaction tube. Amberlyst-15 showed a higher catalytic activity (97% due to its high density of acid
centers
yield [326].21–44%
against It is also worth mentioning
obtained with two others the work of Konrádová
catalysts) et al.,density
due to its high who inof2017aciddeveloped
centers [326].an
interesting
It is also worth protecting-group-free
mentioning the work method starting from
of Konrádová et commercially
al., who in 2017 available aromatic
developed aldehydes
an interesting
and using a microwave
protecting-group-free promoted
method Wittig
starting from reaction of a stabilized
commercially available ylide for thealdehydes
aromatic synthesis and of different
using a
natural products,
microwave promoted including coumarins.
Wittig reaction of a After the optimization
stabilized of the experimental
ylide for the synthesis parameters
of different natural and
products,
the development
including coumarins. of aAfter
general synthetic methodology
the optimization for the parameters
of the experimental synthesis ofand different substitute
the development
coumarin
of a general derivatives
synthetic (Scheme
methodology 12a), for
the the
method was of
synthesis further
differentdeveloped
substituteto incorporate a Claisen
rearrangement
(Scheme 12a), the step. In order
method wastofurther
demonstrate
developed the synthetic utilityaand
to incorporate versatility
Claisen of the developed
rearrangement step. In
order to demonstrate the synthetic utility and versatility of the developed method, the researchbeing
method, the research team performed the total synthesis of two coumarins, one of which team
osthole, a natural
performed the totalcoumarin
synthesis derivative already mentioned
of two coumarins, one of which in being
the anti-inflammatory
osthole, a natural paragraph
coumarin
(Scheme 12b)
derivative [327].mentioned in the anti-inflammatory paragraph (Scheme 12b) [327].
already
O
Ph3P
CHO OCH3
a.
R OH MW (300W), toluene (1.0M) R O O
220°C, 60 min
O O O
O OH Ph3P
H H OCH3
H3CO O O
b. H
TFAA, Cu(acac) H3CO O H2 (1 atm) H3CO O MW (300W), toluene
H3CO OH DBU, CH3CN Pd/CaCO3 185°C, 60 min
-5°C to =àC, 5h EtOAc, r.t.
osthole 78% yield
Scheme 12. Microwave promoted Witting reaction and total synthesis of osthole. (a) general synthetic
methodology; (b) osthole synthesis.
Scheme 12. Microwave promoted Witting reaction and total synthesis of osthole. (a) general synthetic
A broad range of indolo[2,3-c] coumarins have been obtained in good yields (46–88%) by Gu and
methodology; (b) osthole synthesis.
co-workers in 2019 under microwave-assisted base-free intramolecular cross dehydrogenative coupling
using palladium catalyst. The advantages of this methodologies compared to classic synthesis of
A broad range of indolo[2,3-c] coumarins have been obtained in good yields (46-88%) by Gu and
coumarin lactone ring were both atom economy and step efficiency, combined with C-H/C-H coupling
co-workers in 2019 under microwave-assisted base-free intramolecular cross dehydrogenative
without pre-functionalization [328].
coupling using palladium catalyst. The advantages of this methodologies compared to classic
Int. J. Mol. Sci. 2020, 21, 4618 65 of 81
5. Conclusions
The coumarin nucleus has been gaining increasing attention in the last years because of the variety
of its applications both in medicinal chemistry and in agri-food sectors. In this review, we presented
a collection of recent works that highlight the wide range of pharmacological activities ascribable
to coumarins (antioxidant, antibacterial, antifungal, antiviral, anti-proliferative, anti-inflammatory,
antidiabetics, anticoagulant and anti-neurodegenerative) and the possibility to easily modify and
decorate this natural scaffold to perform structure activity relationships studies. The coumarin nucleus
possesses some fundamental properties that ensure it an advisable role in the design of new biologically
active derivatives, mainly due to its stability, low molecular weight and to the easiness to decorate it
for increasing pharmacodynamic and pharmacokinetic properties.
Coumarin scaffold has been a valuable source of inspiration in the design of novel biologically
active compounds and its role as a starting point is very attractive. This natural core is present in a
number of currently available drugs used in the treatment of various diseases. For instance, the use
of warfarin, acenocumarol and phenprocoumon in the treatment and prevention of thromboembolic
diseases is now well established. Other examples include the use of hymecromone as choleretic agent
and that of the antihelminthic haloxon in veterinary medicine. Even though the coumarin nucleus
presents an impressive number of biological activities, its presence among marketed drugs is not
widespread yet. Further efforts are needed in order to achieve coumarin-based compounds with
appreciable pharmacokinetic properties, along with high efficacy and a low toxicity profile.
Moreover, in this review we discussed coumarin photoproperties and their applications as
photocleavable protecting groups and fluorescent probes. Finally, we analyzed the latest updates
in coumarin synthesis from both chemical and technological points of view. One of the most
interesting approaches that emerged from this study is the design and use of coumarin-containing
hybrid compounds.
It is our hope that this review will contribute to further development in the investigation and
exploitation of coumarins’ potential.
Author Contributions: Conceptualization, A.P.; original draft preparation, F.A. and C.P.; writing—review and
editing, S.D., A.P. and L.T.; supervision, A.P. and L.T. All authors have read and agreed to the published version of
the manuscript.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflict of interest.
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International Journal of

Keywords: coumarins; metabolites; biological activity; synthesis; natural sources

Figure 1. Structure of coumarin nucleus.

Int. J. Mol. Sci. 2020, 21, 4618 2 of 81

Figure 1. Structure of coumarin nucleus.

Psoralen (antifungal) [14]

Grandivittin (antibacterial) [16]

Biscoumarins Dicoumarol (anticoagulant) [18]

Phase II metabolism (glucuronide derivative)

In In a recent work, eight coumarin metabolites, which hadbeen

Figure 3. Coumarin structures reported by Couttolenc et al. [30].

Int. J. Mol. Sci. 2020, 21, 4618 7 of 81

5-geranyloxycoumarin (5-GC) 6-geranyloxycoumarin (6-GC)

Figure 7. Structure of theOmostOactive O-prenylated coumarins.

Figure 8. Chemical structure of 5-farnesyloxycoumarin (18) and 8-farnesyloxycoumarin (19).

Figure 9. 4-arylamino-3-nitrocoumarin derivative 20.

Figure 10. Chemical structure of styrylcoumarin 21.

As coumarin nucleus can be widely

Figure 11. General structure of diethylene glycol tethered isatin-1,2,3-triazole-coumarin derivatives

Figure 12. Chemical structure of compound 23.

Figure 13. Chemical structure of umbelliprenin (24).

H H Minimum inhibitory concentration

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 14 of 83

Int. J. Mol. Sci. 2020, 21, 4618 14 of 81

Figure 21. Selected

Figure 24. Coumarin derivatives conjugated to an imidazole-containing moiety.

Figure 24. Coumarin derivatives conjugated to an imidazole-containing moiety.

Coumarin-based 1,4-disubstituted 1,2,3-triazole derivatives.

Figure 27. New prenylated coumarins from Clausena lenis.

Prenylated coumarins came

and 9.13 μg/mL, respectively, against the H1N1 virus.

Figure 30. Hybrid

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 20 of 83

Int. J. Mol. Sci. 2020, 21, 4618 20 of 81

Figure 32. Antiviral coumarins from Bizzarri et al. [129].

Figure 34. Anti-inflammatory

Figure 36. One of the coumarin derivatives isolated from M. exotica.

Figure 38. 7-substituted coumarin as blockers of NF-κB signaling pathway.

Figure 39. Coumarins extracted from Ferulago cassia.

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 26 of 83

Figure 42. Chemical

Figure 43. General structure of compounds 112–117.

Figure 43. General

Figure 45. Chemical

Figure 46. Chemical structure of compounds 148–151.

Figure 47. Chemical structure of compounds 152a–f.

Figure 48. Chemical structure of compounds 153a–e.

Figure 49. Chemical structure of the most common anticoagulant drugs.

Figure 50. Structure of Tecarfarin.

Figure 50. Structure of Tecarfarin.

Figure 51. Chemical structure of coumarins conjugated with salicylic acid.

Structural modifications Chemicalof the 4-hydroxycoumarin

2.11. Antidiabetic Activity

2.11. Antidiabetic Activity

Figure 54. General structures of compounds 158a–e and 159a–e.

Figure 55. Chemical structure of compounds 160–167.

Ahmed A different therapeutic

Figure 57. Chemical structure of compounds 169, 170 and 171.

3. Other Applications of Coumarin Scaffold

3.1. Coumarins Photoproperties

3. Other Applications of Coumarin Scaffold

3.1. Coumarins Photoproperties