4568 Current Medicinal Chemistry, 2011, 18, 4568-4587

Targeting Monoamine Oxidases with Multipotent Ligands: An Emerging Strategy in

the Search of New Drugs Against Neurodegenerative Diseases
L. Pisani, M. Catto, F. Leonetti, O. Nicolotti, A. Stefanachi, F. Campagna and A. Carotti*

Dipartimento Farmacochimico, Università degli Studi di Bari “Aldo Moro”, via Orabona, 4, I-70125, Bari, Italy
Abstract: The socioeconomic burden of multi-factorial pathologies, such as neurodegenerative diseases (NDs), is enormous worldwide.
Unfortunately, no proven disease-modifying therapy is available yet and in most cases (e.g., Alzheimer’s and Parkinson’s disease) the
approved drugs exert only palliative and symptomatic effects. Nowadays, an emerging strategy for the discovery of disease-modifying
drugs is based on the multi-target directed ligand (MTDL) design, an innovative shift from the traditional approach one-drug-one-target
to the more ambitious one-drug-more-targets goal. Herein, we review the discovery strategy, the mechanism of action and the bio-
pharmacological evaluation of multipotent ligands exhibiting monoamine oxidase (MAO) inhibition as the core activity with a potential
for the treatment of NDs. In particular, MAO inhibitors exhibiting additional acetylcholinesterase (AChE) or nitric oxide synthase (NOS)
inhibition, or ion chelation/antioxidant-radical scavenging/anti-inflammatory/A2A receptor antagonist/APP processing modulating
activities have been thoroughly examined.
Keywords: Multi-target directed ligands, Monoamine oxidase inhibition, Acetylcholinesterase inhibition, Neurodegenerative diseases,
Alzheimer’s disease, Parkinson’s disease.

MULTI-TARGET DIRECTED LIGANDS (MTDLs)
Largely spread pathologies such as neurodegenerative diseases
(NDs), diabetes, cardiovascular diseases and cancer [1], share a
common multi-factorial nature. They display complex and not fully
understood onset and progression implying the deregulation of
multiple, and often unrelated, cellular physiological events that are
caused by genetic, environmental and endogenous pathogenic
factors. The complexity of this biochemical landscape suggests that
the targeting of a single pathophysiological process among the array
of the altered ones may be a challenging goal but it will likely result
largely inadequate to prevent, retard, halt or reverse the disease.
Over the years, deep efforts have been made in order to discover the
key biochemical events triggering these diseases and to identify the
most crucial druggable targets. Indeed, many new drugs showing a
single–target mechanism have been discovered and are currently
used in many multi-factorial pathologies although their therapeutic
efficacy is limited to an essentially palliative and/or symptomatic
effect. Hence, the discovery of genuine disease-modifying drugs
addressing a multi-factorial disease through a single-targeting
action can be considered as a limited and generally losing strategy.
The failure of the so-called one-drug-one-target paradigm
urged researchers to investigate alternative ways to manage more
efficiently diseases triggered by the combination of several cellular
aberrations and suggested a new and different approach by
addressing simultaneously diverse biological targets that operate
independently or cooperatively [2]. This approach might be applied
by using a combination of two or more drugs (polypharmacology),
each targeting a single biochemical process, or a single molecular
entity able to exert beneficial therapeutic effects by modulating
different targets.
So far, the polypharmacology approach has represented the
most exploited way to tackle multi-factorial diseases and two main
strategies have emerged in this field. As a first case, a cocktail of
two or more drugs addressing different biochemical targets have
been used following appropriate therapeutic protocols. The main
problems associated with this approach reside in the low patient
compliance and adherence, mostly due to the daily assumption of a
high number of drugs often with repeated and varying doses, and in
the high chance of drug-drug interactions and multiple competing
metabolic transformations. To overcome these drawbacks, a second
strategy that combines different drugs with a well defined dose ratio
*Address correspondence to this author at the Dipartimento Farmacochimico,
Università degli Studi di Bari “Aldo Moro”, via Orabona, 4, I-70125, Bari, Italy; Tel:
+390805442782; Fax: +390805442230; E-mail: carotti@farmchim.uniba.it
within a unique formulation has been envisaged. Despite both
patient compliance and administration regimen may result
improved, some disadvantages may arise from undesired drug-drug
interactions, extremely high variability of patient responses and an
unforeseeable toxicological profile. Within this frame, an emerging
strategy is now coming to the attention of the medical and
pharmaceutical communities to improve the efficacy of the
therapeutic protocol and enhance the drug safety. This alternative
strategy is grounded on the assumption that a single, properly
designed molecular entity, may be able to interfere with different
cellular events and cope with a multi-factorial disease more
efficiently [3]. The combination of different biochemical and
pharmacological activities in a single molecule (multi-target
directed ligand, MTDL) [4] may provide a series of benefits mainly
related to unique pharmacokinetic and pharmacodynamic patterns
in comparison to drugs cocktails. Indeed, the pharmacokinetic and
clinical profiling of a multipotent drug is greatly made easier, being
limited to the study of ADME (adsorption, distribution, metabolism
and excretion) and toxicological properties of a single molecular
entity. Moreover, the use of a multipotent drug will clearly simplify
the administration regimen, improve the patient compliance, and
avoid the risk of adverse drug-drug interactions.
Basically, the multiple action of a drug could be unveiled by
serendipitous observations and by the repositioning study of old
drugs, that is a systematic search of potential additional activities of
already-known molecules often revealing an unexpectedly wider
pharmacological domain [5]. Next, the discovery of multipotent
molecules can be pursued through a knowledge-based or a library
screening approach [6]. In the former, the starting point is
represented by a bioactive compound already biased towards a
specific target of interest. Typically, a series of rational structural
modifications are introduced to add further desired activities to the
original one. The latter discovery method relies on the sequential
screenings of selected molecular libraries on the multiple targets of
interest. These libraries are preliminarily screened to shortlist
compounds having the most desired/important biological activity.
The filtered compounds are then subject to cross-screening towards
other possible targets under investigation. Normally, the campaigns
of wet screening are anticipated by a cheaper virtual screening
performed by using as a query a multi-pharmacophore model
integrating the common structural features shared by the
pharmacophore models developed for each single target.
When a more traditional, but very rational design strategy is
applied, the molecular framework of a multi-target molecule might
be built up with three different approaches. One classical way
consists in the molecular heterodimerization of two structurally

0929-8673/11 $58.00+.00 © 2011 Bentham Science Publishers

Targeting Monoamine Oxidases with Multipotent Ligands Current Medicinal Chemistry, 2011 Vol. 18, No. 30 4569

C F H
O U Y
N S B
J I R
U O I
G N D
A I
T Z
I A
O T
N I
O
N

LINKER

A B C

Fig. (1). Rational design strategies to obtain multi-target ligands.

diverse scaffolds, each responsible for a different biological action.
These molecules are structurally joined (conjugated) through an
appropriate linker (compound A, Fig. 1), that can be either
metabolically stable or cleaved to release the two active moieties
(dual pro-drugs).
In a second method, the dual activity might be exhibited in the
absence of a linker, by a compound resulting from the “exact
fusion” (e.g., through a single bond connection) in a single
molecular entity of two molecules with different biological
activities (compound B, Fig. 1). A third strategy is based on the
preliminary identification of the key structural fragments
(pharmacophoric features) responsible for a defined biochemical
activity in different molecules. Then these structural elements are
combined in a novel unique, appropriately designed, molecular
hybrid (compound C, Fig. 1) [7]. The latter is probably the most
exploited and feasible design method that can be applied when the
two sets of “active” molecular fragments are easily identifiable and
the structural similarity between the starting compounds is
relatively high.
In most cases, the potential multi-target hit/lead compounds
coming out from all of these discovery strategies need further
optimization to suitably balance their in vitro and in vivo
pharmacological properties.
Monoamine Oxidases (MAOs): A “Druggable” Target in
Neurodegenerative Diseases
Monoamine oxidase (MAO, EC 1.4.3.4, amine-oxygen
oxidoreductase) is a flavoenzyme localized at the inner side of the
outer mitochondrial membrane to which is tightly bound through its
C-terminal sequence. This enzyme is ubiquitously distributed in
nature, both in human and animal tissues. Its biochemical function
resides in the oxidative deamination of xenobiotic [8] and
endogenous amines, including monoamine neurotransmitters such
as serotonin (5-HT), noradrenaline (NA), and dopamine (DA) [9].
Two distinct mechanisms have been postulated for MAO-A
degradation both leading to an iminium intermediate that is further
hydrolyzed to the final product, that is an aldehyde. As depicted in
Fig. (2), one process is based on a single electron transfer (SET)
whereas the other involves a polar nucleophilic attack of the
degradation-prone amine to the flavin cofactor (Fl, Fig. 2) [10].
Two distinct enzymatic isoforms, namely MAO-A and MAO-
B, have been isolated and fully studied. They are characterized by
different amino acid sequences, three-dimensional structures [9],
organ and tissue distributions [11], sensitivity to inhibitors [12] and
substrate specificity. Both enzymes oxidatively deaminate DA,
whereas MAO-A preferentially deaminates 5-HT, adrenaline and
NA and is selectively inhibited by clorgyline. MAO-B
preferentially metabolizes benzylamine and
-phenethylamine
(PEA) and is inhibited by selegiline and rasagiline selectively.
Isoform ratio and tissue distribution of MAOs may vary according
to the species. Thus human and rat liver express almost identical
levels of both MAO isoforms. MAO-A predominates in human
placenta and intestinal mucosa [13], whereas MAO-B is the
prevalent isoenzyme found in platelets [14]. Both isoforms are
present in rat and human brain, with proportions depending on the
specific neural region [15]. Adrenergic and histaminergic neurons
contain prevalently MAO-A and MAO-B isoforms, respectively.
4570 Current Medicinal Chemistry, 2011 Vol. 18, No. 30 Pisani et al.

AMINIUM CATION RADICAL MECHANISM

Flox Fl Fl Flred
-H H2O
R CH2 NH2 R CH2 NH2 R CH NH2 R CH NH2 R CHO

POLAR NUCLEOPHILIC MECHANISM

Flox -H H2O
R CH2 NH2 R CH2 NH Fl R CH NH2 + Flred R CHO
Basic
aminoacid

Fig. (2). Proposed mechanisms of oxidative deamination catalyzed by MAO-A [10].

Taking into consideration the isoforms differences, the native role
of MAOs, and their consequent implication in the catabolic
degradation of many biogenic amine neurotransmitters, these
enzymes have been regarded as attractive targets for the treatment
of widespread neurological disorders [16]. Several selective MAO-
A inhibitors exhibited antidepressant activity (e.g., moclobemide,
brofaromine, clorgyline and toloxatone, Chart 1) [17]. whereas
selective MAO-B inhibitors have been introduced in the therapy of
Parkinson’s disease [18] (PD) (e.g., lazabemide, selegiline, and
rasagiline, Chart 1). Iproniazid, originally used as an anti-
tuberculosis agent, was the earliest unselective MAO inhibitor
introduced in the therapy of depressive disorders in the 1950s and
later withdrawn because of hepatotoxic side-effects. Other agents
acting as MAO inhibitors (MAO-Is) that still exhibited a non-
selective inhibition of both isoenzymes (phenelzine, isocarboxazid,
and tranylcypromine, Chart 1) have been later introduced. The
major drawbacks linked to these antidepressants were represented
by severe side-effects such as the hepatotoxicity and the
hypertensive crisis triggered by the consumption of tyramine-rich
food (e.g., wine, beer and especially cheese and therefore called
“cheese-effect”). Since 80% of intestinal MAOs is constituted by
the A isoform, the block of this enzyme is responsible for the halted
degradation of tyramine and the “cheese effect” can be therefore
ascribed to the lack of selectivity of the earliest MAO-Is. As a
result, the clinical use of MAO-Is as antidepressants has been
progressively reduced because they required severe dietary
restrictions and safer drugs such as tricyclic antidepressants and
selective serotonin reuptake inhibitors (SSRIs) have been preferred.
Currently, MAO-Is constitute the third- or fourth-line treatment of
depression [19].
The inhibition of MAOs still represents an interesting field of
research thanks to some new emerging and promising therapeutic
perspectives. In fact, the ongoing medicinal chemistry research of
selective and reversible MAO-A inhibitors (RIMA) with a better
therapeutic profile, fewer drug-drug interaction, and lower adverse
effects is encouraged by solid evidence enlightening the
effectiveness of MAO-Is in the treatment of major depression,
treatment-resistant major depressive disorder and bipolar
depression [19]. Moreover, MAO-B inhibitors gained also new
consensus as safer drugs for the treatment of early-phase PD,
providing symptomatic relief of motor deficits and retarding the
need of levodopa administration. Interestingly, they exhibited a
wide tolerability and may perform a disease-modifying action in the
long-term treatment of PD [20].
In addition, a very recent and renewed interest has regarded
MAOs as potential targets in other NDs such as Alzheimer’s and
Huntington’s disease (AD and HD, respectively), and amyotrophic

O
H
H H O N
N N N NH2
NH2 N N H
H N
O

Phenelzine (NS) Isocarboxazid (NS) Iproniazid (NS) Tranylcypromine (NS)

H
N OH
O O Cl

N O O N
N
O N
H
O
Cl Cl
O

Br
Brofaromine (A) Moclobemide (A) Toloxatone (A) Clorgyline (A)

O HN
NH2 N
N
H
N
Cl
Lazabemide (B) Selegiline (B) Rasagiline (B)

Chart 1. Chemical structures of non-selective (NS) and selective MAO-A (A) and MAO-B (B) inhibitors.
Targeting Monoamine Oxidases with Multipotent Ligands
lateral sclerosis (ALS). Within the complex biochemical portrait
compromising neuronal viability in these pathologies, some cellular
impairments may be ascribed to the enzymatic activity and peculiar
mechanism of action of MAO. The enzymatic action of MAOs sets
the reduction of neurotransmitter levels in the synaptic cleft, thus
altering the signalling transfer mechanisms. Moreover, as a result of
the oxidative deamination of neurotransmitters and xenobiotic
amines, hydrogen peroxide (H2O2) is produced. H2O2 represents a
strong oxidant itself and is a precursor of radical reactive oxygen
species (ROS), that can promote harmful cellular alterations in
pathological conditions. Hydrogen peroxide generated through
MAO catalytic cycle can interact with free Fe2+ cation in the Fenton
reaction (Scheme 1), resulting in the production of ROS and
neuronal oxidative stress. Therefore, MAO activity is thought to
play a key role to the onset of oxidative stress. In physiological
conditions, ROS, e.g., superoxide radical anion (·O2-), hydroxyl
radical (·OH), and hydrogen peroxide are produced through several
processes including mitochondrial respiration, cellular metabolism,
and degradation of dietary components. Cellular ROS levels are
balanced by the action of an endogenous detoxification system
involving glutathione (GSH) and several enzymes, namely
glutathione reductase (GRD) and glutathione peroxidase (GPX),
catalase (CAT) and superoxide dismutase (SOD). These redox
systems are responsible for the transformation of these reactive
species in non-toxic compounds. When the amount of ROS is
extensively increased and/or the efficacy of this protective redox
system is impaired in many pathological conditions, ROS
production becomes potentially harmful. Oxidative radicals may
react with proteins and DNA, producing base-pair mutations,
deletions and insertions [21], and cause lipid peroxidation, thus
affecting cell membrane integrity. Ultimately, these biochemical
aberrations induced by ROS lead to cellular damage and death.
MAO
oxidation
dopamine
biosynthesis
Fe 2+ -OH
Fe 2+
Fenion reaction
dopamine H2O2
autoxidation
GSH GRD
SOD
GSSG
GPX
CAT
-O2+ H2O
respiration
O2
Scheme 1. ROS endogenous production and detoxification system.
For all these reasons, MAO may be considered one of the most
interesting potential target in the design of multipotent ligands that
might exhibit a disease-modifying activity towards multi-factorial
NDs.
The extension of life expectancy along with the progressive
increase of elderly population and improvements of health care
claim for a great attention towards age-related NDs (e.g. AD, PD,
HD and ALS). Each pathology possesses its characteristic
symptomatic manifestations, histological hallmarks and
biochemical mechanisms. Nevertheless, NDs share some common
and general impairments ultimately leading to neuronal death
and/or loss of neuronal functionality. Among others, oxidative
Current Medicinal Chemistry, 2011 Vol. 18, No. 30 4571
stress conditions [22], biometals imbalance [23], protein misfolding
and aggregation [24], and mitochondrial damage [25] play a crucial
role.
The scope of the present review is the collection and discussion
of the main results of the research projects focused on the discovery
of multipotent ligands showing MAO inhibitory activity as a key
biochemical feature. Since MAO inhibition occupies a central role
in neurodegeneration, we paid attention to multi-target molecules
with a therapeutic potential for AD, PD, HD and ALS. From a
literature survey, in contrast with other targets (e.g., AChE) MAO
inhibition via multi-target ligands has not extensively attracted the
attention of medicinal chemists although convincing experimental
evidence demonstrated the pivotal role of these enzymes in NDs.
To date, MTDL strategy targeting MAOs among other targets
represents an elegant and promising strategy even if it has not led
yet to novel clinical therapeutics, with the exception of rasagiline
whose multi-targeting profile has been discovered later.
Interestingly, some drug candidates emerged from MTDL design
showed promising multi-target properties and have been submitted
to extensive bio-pharmacological profiling, as described in the
present survey.
Rasagiline as a Multipotent MAO Inhibitor
Rasagiline (1) (N-propargyl-1-(R)-(+)-aminoindan) [26], the R-
isomer of a molecule originally coded as AGN1135, is a potent,
highly selective and irreversible MAO-B inhibitor which has been
developed as an anti-Parkinson drug in monotherapy or as an
adjuvant drug in levodopa treatment [27]. In comparison to
selegiline (2), that is another potent, selective and irreversible
“suicide-type” MAO-B inhibitor, the pharmacokinetic profile of
rasagiline presents safer features since it is not linked to the in vivo
metabolic production of L-amphetamine and L-methamphetamine
[28] responsible for some sympathomimetic adverse effects such as
increased blood pressure and heart rate [29]. In addition, rasagiline
shows a neuroprotective and anti-apoptotic action in PC-12 and
neuroblastoma SH-SY5Y cellular lines against different
neurotoxins such as SIN-1 (a peroxynitrite donor) [30], MPTP [31],
6-hydroxydopamine (6-OHDA) [32], and N-methyl-(R)-salsolinol
[33] and protects from neuronal damage caused by several insults in
vivo, e.g., global ischemia, anoxia and head injury [34].
N
HN
Rasagiline (1) Selegiline (2)
rat brain: rat brain:
MAO-A IC50 = 412 nM MAO-A IC50 = 944 nM
MAO-B IC50 = 4.43 nM MAO-B IC50 = 3.63 nM
human: human:
MAO-A IC50 = 710 nM MAO-A IC50 = 1700 nM
MAO-B IC50 = 14.0 nM MAO-B IC50 = 6.80 nM
Fig. (3). MAOs inhibitory activities of rasagiline (1) and selegiline (2) [35].
Strong evidence supports the idea that this neuroprotection is
not related to the inhibition of MAO enzymatic activity. As a matter
of fact, the S-isomer of rasagiline, called TVP1022, differs from
rasagiline being a 2000-fold weaker human MAO-B inhibitor (IC50
equal to 28.7 versus 0.014 μM) [35] but holds comparable
neuroprotective activity. Therefore, this action has been ascribed to
4572 Current Medicinal Chemistry, 2011 Vol. 18, No. 30
the presence of the reactive propargylamino moiety which might
interfere with many other cellular processes [36]. It has been
demonstrated that rasagiline acts on different key steps of the
apoptotic cascade. In fact, 1 up-regulates the expression of
antiapoptotic proteins Bcl-2 and Bcl-xl, belonging to Bcl-2 family,
while down-regulates Bad and Bax [37], through the stimulation of
PKC
and  and inhibition of pro-apoptotic PKC and . Moreover,
rasagiline prevents the decline in mitochondrial membrane
potential, the activation of caspase 3, and the nuclear translocation
of glyceraldehyde-3-phosphate dehydrogenase [38]. The
consequence is the block of both the mitochondrial swelling
process and the opening of the permeability transition pore, that are
two cellular events that initiate the apoptotic cascade. Interestingly,
rasagiline and some related propargylamines, promote the non-
amyloidogenic processing of amyloid precursor protein (APP) by
stimulating the PKC and MAP-kinase mediated cleavage operated
by
-secretase [37c, 39]. This proteolytic pathway produces soluble
fragments of APP (sAPP) that are unable to initiate the amyloid
cascade leading to the neurotoxic fibrillogenesis [40].
Furthermore, ADME studies of rasagiline highlighted the role
of its major hepatic metabolite, the 1-(R)-aminoindan, which is still
a weak and reversible MAO inhibitor endowed with
neuroprotective properties that could contribute to the overall
biological effects observed for rasagiline [41].
Taken together, these findings lead to the conclusion that
rasagiline could be considered a highly valuable drug against PD
not only for a symptomatic dopamine-replacement therapy but also
for a promising disease-modifying profile of the severe motor and
neurological deficiencies of PD [27a, 42].
Dual MAO-Acetylcholinesterase (AChE) Inhibitors
Alzheimer’s disease (AD) is the most diffuse form of senile
dementia and represents the fourth leading cause of death in
western countries. More than one century has passed from the
discovery of this disease by the German neuropsychiatrist Alois
Alzheimer [43] and different studies have been undertaken to shed
light into the pathophysiological mechanisms underlying this
devastating neurological disorder. Notwithstanding, a complete
comprehension of the origin and progression of AD is far to be
reached. What is clear is the fact that AD is a complex disease
triggered by several cellular impairments regarding, among many
others, cholinergic neurons deficit in the basal forebrain ultimately
leading to neuronal loss. From a histopathological viewpoint,
convincing studies identified some unequivocal hallmarks of AD: i)
extracellular fibrillary aggregates of -amyloid (A) peptide (senile
N
O N
N
NH2
Tacrine (Cognex®) Rivastigmine (Exelon®)
OH
O N
O
O
N
O
Galantamine (Reminyl®)
Chart 2. Approved drugs for AD.
Pisani et al.
plaques), soluble peptidic fragments mainly located in neocortical
regions and derived from sequential - and -secretase proteolytic
cleavage of APP; and ii) intracellular neurofibrillary tangles (NFT),
paired helical structures mainly constituted of insoluble
hyperphosphorylated
protein, accumulated in the medial temporal
lobe [44]. These biochemical hallmarks are associated with an
increased oxidative stress, abnormalities in endoplasmic reticulum
and deficiencies in the clearance process operated by the ubiquitine-
proteasome system (UPS). Both A oligomers and
self-aggregates
are cytotoxic so that oxidative and inflammatory insults seem to be
a direct consequence of the misfolding of these proteins in neuronal
cells. AD is characterized by the progressive loss of brain capacities
including memory loss, learning and language difficulties, and
diminished simple problem-solving capabilities mainly controlled
by the cholinergic neurotransmission. In basal forebrain cholinergic
neurons of late-stage AD patients, it has been documented a
dramatically reduced level of neurotrophins, that manage learning,
memory and behaviour in physiological conditions [44]. On this
ground, the so-called “cholinergic hypothesis” has been proposed
[45] which suggested the enhancement of the acetylcholine (ACh)
concentration and/or of its action in the CNS, as a viable and
winning strategy to solve cognitive and behavioural symptoms. In
principle, this goal may be attained throughout the use of AChE-Is
[46] or sub-type selective muscarinic agonists [47].
An increased potential efficacy of some AChE-Is stems from
the occasional discovery of a non-catalytic action of the enzyme,
whose peripheral anionic subsite (PAS) proved to be the structural
motif involved in the promotion of the aggregation of A to form
the amyloid plaques [48].
Nowadays, inhibition of AChE represents the predominant
pharmacological approach that has led to the development of anti-
Alzheimer drugs. Three AChE-Is (Chart 2), i.e., donepezil,
rivastigmine and galantamine – the hepatotoxic tacrine was
withdrawn from the market – are currently used in clinical practice
in the moderate form of AD but their therapeutic efficacy is still
under debate, since they act symptomatically in relieving the AD-
related cognitive decline by restoring adequate levels of ACh
without affecting the leading causes of cholinergic neuronal loss
[49]. Another drug, memantine, with a different mechanism of
action (NMDA antagonism) and a limited efficacy is now available
to treat AD in a more advanced stage.
Behind the central role of cholinergic transmission, the
complexity of AD advocated many other triggering factors of the
neurodegenerative process. One of them is the “amyloid cascade”,
whose study has led to a deeper comprehension of the pathology of
O
NH2
Memantine (Namenda®)
O
Donepezil (Aricept®)
Targeting Monoamine Oxidases with Multipotent Ligands
R2
R1
R1 = R2 = H;
R3 = R4 = Me
AChE
MAO-A IC50 = 0.92 μM
MAO-B IC50 = 317 μM
R1
O
(3b) (3c)
R1 = NHMe; R2 = H R1 = NHMe; R2 = H;
R3 = R4 = Me R3 = Me; R4 = Et
AChE IC50 = 3.36 μM AChE IC50 = 5.68 μM
MAO-A IC50 = 13.6 μM MAO-A IC50 = 3.74 μM
MAO-B IC50 = >1000 μM MAO-B IC50 = >1000 μM
Fig. (4). Tricyclic derivatives 3 with dual MAO-AChE inhibitory activity [59].
AD and has provided insights for the design of new potential
therapeutic agents [50]. According to this hypothesis, the increase
of A
production and the aggregation into low-molecular weight
oligomers, protofibrils, fibrils and ultimately amyloid plaques, are
the leading causes of the onset and progression of AD. The
reduction of both A
formation (with
- and
-secretases
modulators) [51, 52] and aggregation (with anti-aggregating agents)
[53], and the increase of the A
clearance (with active and passive
immunization [54]) are potential therapeutic strategies for the
therapy of AD. Recent studies enlightened also the role of oxidative
stress [55], metal ion dyshomeostasis, and inflammation in AD
pathogenesis. Neuronal death is the result of different insults,
comprising bio-macromolecules alteration upon peroxidation
reactions. Oxidative damage may arise from MAO-induced free
radicals, mitochondrial abnormalities, inflammation, or redox active
metals, that can contribute to the excessive production of toxic ROS
through Fenton chemistry.
Within this complex scenario, MAO-Is might play a beneficial
neuroprotective action in NDs by reducing oxidative stress
conditions [27d, 56]. The following experimental evidences support
this research line: i) up to 3-fold increased MAO-B activity in the
temporal, parietal and frontal cortex has been observed in patients
suffering from AD compared to control healthy patients [57]; ii) the
connection between behavioural disorders and the observed
modification in other neurotransmitter systems is mediated by
biogenic amines (e.g., serotonin, noradrenaline) whose level is
influenced by the catabolic action of MAOs.
The high complexity of AD calls for a huge effort to find
molecules able to modulate multiple CNS targets and then to
promote a real improvement of AD therapy. The multi-factorial
nature of the disease rules out the use of consolidated mono-therapy
and put researchers on the way of multipotent drugs hitting
cooperatively different targets underlying the onset and/or
progression of the disease. This approach is grounded on the idea
that an AChE-I could exhibit a more efficient therapeutic effect in
AD when able to express an additional MAO inhibitory activity,
limiting the sources of ROS-related neurotoxicity [58].
As already mentioned, one of the most exploited way to obtain
a MTDL is to combine in a unique molecular framework different
pharmacophoric features responsible for well-defined and different
Current Medicinal Chemistry, 2011 Vol. 18, No. 30 4573
N N R4
R3
(3a)
n.d.
R2
N N R4
R3
(3d) (3e)
R1 = NHMe; R2 = Br; R1 = NMe2; R2 = Br;
R3 = Me; R4 = Et R3 = R4 = Me
AChE IC50 = 0.14 μM AChE IC50 = 0.060 μM
MAO-A IC50 = 0.21 μM MAO-A IC50 = 0.54 μM
MAO-B IC50 = >1000 μM MAO-B IC50 = 51.0 μM
biological actions. Hopefully, the resulting hybrid molecules will
retain the biological activities associated to each different
pharmacophore and exert beneficial effects from the synergy of
these combined mechanisms.
A pioneering work in the field of hybrid multi-target anti-AD
drugs was carried out by D. M. Fink and M. G. Palermo [59]. They
designed dual MAO-AChE inhibitors by decorating a tricyclic
1,2,3,4-tetrahydrocyclopent[b]indole carbamate scaffold, inspired
by the known AChE-I physostigmine, with a propargylamino
group, a recurrent pharmacophoric group of different MAO-Is.
The interest for this molecular series might be limited by their
irreversible and selective MAO-A inhibition due to the presence of
the reactive N-propargylamino group, that is likely associated to
adverse side-effects [60]. More interestingly, the synthetic iminic
intermediates (3a-3e) were also tested and showed from moderate
(3b-3c) to good (3d-3e) dual inhibitory activities. Regarding MAO
inhibition, 3b-3e exhibited a reversible mechanism of action and a
marked selectivity for MAO-A. Thus structure-affinity and
structure-selectivity relationships, SARs and SSRs, respectively,
were examined for these derivatives. The introduction of the
carbamate moiety induced a weak affinity for AChE while reducing
MAO-A inhibition not so drastically (compare 3a and 3b, Fig. 4). A
critical influence was exerted by the R4 substituent at the iminic
nitrogen. Increasing the size of R4 alkyl groups had a slighter
influence over AChE than over MAO inhibitory potency (compare
3b and 3c), the ethyl group being the best substituent to maintain a
good dual activity as proved by derivative 3c. The R2 substituent
ortho to the carbamate moiety affected both enzymatic activities
leading to the very interesting compounds 3d-3e. Unfortunately, a
further development of these compounds was halted by the low oral
activity and poor brain penetration found during ex vivo studies on
brain tissues.
Starting from the observation that ensaculin, a compound
bearing a coumarin skeleton, showed an interesting AChE
inhibition in vitro (IC50 = 0.36 μM) [61], a set of various 7-
substituted coumarin derivatives (Fig. 5), previously synthesized
and characterized as potent MAO inhibitors [62], was screened by
some of us on AChE. All the tested derivatives inhibited AChE
with Ki values in the medium to low micromolar range (3-100 μM)
[63]. As for MAO inhibition, they were endowed with micromolar
4574 Current Medicinal Chemistry, 2011 Vol. 18, No. 30 Pisani et al.

N R1
O N O
R2
O O O
O O O
Ensaculin (4a-g)

Fig. (5). Ensaculin and coumarin derivatives 4 with dual MAO-AChE inhibitory activity.

Table 1. MAO and AChE Inhibition Data of Coumarin Derivatives 4a-g [62]

AChEa MAO-Ab MAO-Bb

Compd R1 R2
(Ki) (IC50) (IC50)

4a Me H 100 μM 35%c 18%c

4b Me Bn 4.07 μM 0.69 μM 4.37 nM
4c H Bn 30.9 μM 1.95 μM 18.2 nM
4d Me (3’-Me)Bn 9.33 μM 3.31 μM 4.37 nM
4e Me (3’-OMe)Bn 17.8 μM 1.51 μM 3.63 nM
4f Me (3’-F)Bn 7.76 μM 0.58 μM 2.82 nM
4g Me (3’-Cl)Bn 3.39 μM 1.12 μM 3.31 nM
a
Electric eel AChE (EC 3.1.1.7) was used. Rat brain mitochondrial suspensions were used. Percentage of inhibition at [inhibitor] = 30 μM.
b c

to low nanomolar affinities and a marked selectivity for the B
isoenzyme. The benzyloxy group linked to C7 of coumarin ring was
the main molecular determinant of the high MAO-B affinity and its
absence reduced, albeit less drastically, the AChE inhibition (see
compound 4a in Table 1). To ascertain the mechanism of AChE
inhibition by compounds 4, a kinetic analysis of selected 7-
benzyloxy coumarins was undertaken. Lineweaver-Burk and Hanes
plots highlighted the non-competitive character of the AChE
inhibition, that could be of great interest in the context of
Alzheimer’s disease as this kinetic mechanism may be ascribed to
the interaction of the inhibitor at the PAS. This kinetic behaviour
was later confirmed on a series of potent and selective
heterodimeric AChE-Is containing the coumarin ring [64]. The
coumarin nucleus resulted optimal for the dual AChE-MAO affinity
and its replacement with a 4H-chromen-4-one reduced both
enzymatic inhibitory potencies. The introduction of an additional
methyl group at position 3 of the coumarin ring increased both
MAO and AChE affinity (compare 4b and 4c in Table 1). Focused
SARs on the benzyloxy substitution pattern revealed that
substituents in meta position decreased the AChE affinity
(compounds 4d-4f) with the notable exception of the m-chloro
substituent (compound 4g), that resulted the most potent AChE
inhibitor of the whole series and exhibited outstanding MAO-B
affinity and selectivity over MAO-A. Compound 4g would deserve
further optimization and investigation as a potential anti-Alzheimer
agent.
Youdim and coll. studied a very large series of N-
propargylaminoindans and N-propargyl-phenethylamines (Fig. 6) as
dual MAO-AChE inhibitors [65]. The molecular design was based
on the combination in the same molecule of a propargylaminic
fragment, characterising well known MAO-Is such as rasagiline and
selegiline, with a carbamate moiety, which could add the AChE
inhibitory property. In addition to AChE and MAO-B inhibition
these molecules might display a potential antidepressant action,
through MAO-A inhibition, and induce a neuroprotective effect
unrelated to MAO inhibition, due to the presence of the
propargylamino group. MAO-A inhibition showed by derivatives 5-
6 constitutes only an apparent drawback linked to possible
tyramine-potentiation because a stronger and more specific
inhibition of central with respect to the peripheral (liver and
intestine) MAO-A was claimed [66]. In this research project, two
hit compounds belonging to the indan family (5a-b) have been
identified along with an additional candidate bearing a
phenethylamine system (6). Ladostigil (5b, TV3326) has
comparable equine butyrylcholinesterase (BChE) and human AChE

R4
O R3 R1
R3 N O N
N O R4
R2 N O R2
R1
(5a) Ladostigil (5b) (6)
R1 = H; R2 = Me; R1 = H; R2 = Me; R1 = R2 = R3 = Me;
R3 = n-Pr; R4 = Cl R3 = Et; R4 = H R4 = n-Hexyl

AChE IC50 = 43.9 μM AChE IC50 = 52.4 μM AChE IC50 = 3.06 μM

MAO-A IC50 = 375 μM MAO-A IC50 = 85.0 μM BChE IC50 = 0.72 μM
MAO-B IC50 = 32.0 μM MAO-B IC50 = 120 μM MAO-A IC50 = 20.0 μM
MAO-B IC50 = 1.50 μM

Fig. (6). Carbamate derivatives 5-6 with dual MAO-AChE inhibition activity [65].
Targeting Monoamine Oxidases with Multipotent Ligands
inhibitory activity, with a greater potency against the former and a
duration of action longer than rivastigmine. At a preliminary stage,
the introduction of the carbamate moiety resulted in the loss of
MAO inhibitory activity, as assessed by in vitro assays on rat brain
homogenates. Notwithstanding, ladostigil, submitted to in vivo
studies showed a dose-dependent and not selective MAO-A/-B
inhibition in rats, mice and monkeys after chronic oral
administration (25–150 mmol/kg dose once daily, for 14 days) [67].
Fortunately, MAO-A inhibition activity was markedly less
pronounced in liver and small intestine than in CNS and this result
was attributed to the action of local active metabolites in the CNS.
The 6-hydroxy derivative, produced by the metabolic degradation
of the carbamic group, has weak in vitro activities (IC50 against
MAO-A and B of 0.46 mM and 0.35 mM, respectively). Thanks to
these central effects, ladostigil is able to elevate dopamine,
noradrenaline and serotonin levels and these synergic actions result
in a promising antidepressant profile. Moreover, by lacking
peripheral activity on both MAO isoforms, this molecule is devoid
of the cardiovascular side-effects associated with tyramine-induced
“cheese-effect”. Ladostigil prevents MPTP-induced parkinsonism
in mice model [68], as a typical MAO-B inhibitor, and shows
neuroprotective functions similar to those of rasagiline in several in
vitro [69] and in vivo models [70], likely due to the presence of the
propargylaminic moiety. As demonstrated for the well-known
propargylaminic MAO-Is selegiline and rasagiline, additional
neuroprotective mechanisms might be exerted through the block of
cellular death signalling process and the production of different
neurotrophic factors, namely neurotrophins and ligands of glial cell
line-derived neurotrophic factor [71]. Further investigations for
these novel classes of multipotent molecules are warranted to meet
the following goals: (a) closer AChE and MAO-B affinities, (b)
good to moderate AChE inhibition and (c) improved MAO-B
affinity and MAO-B over MAO-A selectivity.
R2
N
N S
NH
S
R1
(7a) (7b)
R1 = Me; R2 = OMe R1 = Et; R2 = OMe
AChE IC50 = 0.090 μM AChE IC50 = 2.55 μM
MAO-A IC50 = 450 μM MAO-A IC50 = 421 μM
MAO-B IC50 = 43.0 μM MAO-B IC50 = 22.0 μM
Fig. (7). Thiocarbamoyl-3-phenyl-5-thienyl-2-pyrazoline derivatives 7 with
dual MAO-AChE inhibitory activity [73].
Aiming at the discovery of new drugs with multiple actions able
to perform a disease-modifying effect in NDs, Gökhan and co-
workers evaluated a series of racemic 1-N-substituted-
thiocarbamoyl-3-phenyl-5-thienyl-2-pyrazoline derivatives (Fig. 7),
previously investigated as MAO-Is [72], for their ability to block
ChEs activity. Their research project was based on the assumption
that AChE inhibitory properties might be conferred to these
molecules by the thiocarbamoyl moiety, supposedly acting as an
isosteric substitution of a carbamic functionality [73]. The analysis
of SARs for this small series of congeners indicated that the
electron-releasing p-methoxy group in the phenyl ring plays a
crucial role in enhancing both affinity and selectivity towards
AChE and MAO-B compared to BChE and MAO-A, respectively.
Some of these inhibitors were reported as moderate and selective
MAO-BIs with an IC50 ranging from 22 to 92 μM on bovine liver
homogenates assays but they displayed an irreversible-type
Current Medicinal Chemistry, 2011 Vol. 18, No. 30 4575
mechanism of action. Interestingly, these N-substituted pyrazolines
were also active against human erythrocyte and plasma AChE and
exhibited a selective, reversible and non-competitive inhibition
profile. The comparison of compounds 7a and 7b shows that AChE
affinity was notably decreased by the increasing size of the R 1
substituent of the thiocarbamoyl exocyclic nitrogen. The optimal
substitution of the phenyl ring and the thioureidic moiety,
represented by a p-methoxy and a methyl group, respectively,
furnished a promising derivative (7a) exhibiting a strong AChE
affinity with an IC50 in the nanomolar range and a moderate MAO-
B inhibitory potency and selectivity.
Despite the outstanding innovations in synthetic organic
chemistry that could have expedited the preparation of new
bioactive compounds, nature still remains a great source of
structurally diverse and promising molecules for the drug discovery
process. To perform a medium/high-throughput screening of
naturally-derived products, an efficient microtitre plate method
based on Ellman’s assay was developed and validated for the study
of both electric eel AChE inhibition and kinetic behaviour [74].
When this microplate technique was applied to the biological
screening of a library of 45 non-alkaloidal natural compounds in the
search of novel AChE-Is, several xanthones were identified as good
to moderate inhibitors. Four of them (8a-c and 9, Fig. 8) are worth
noting because previous studies had highlighted their promising
MAO affinity [74b], thus representing dual AChE-MAO ligands.
Electron-absorption spectroscopy revealed that the mechanism of
these reversible MAO-Is is likely mediated by the formation of
charge-transfer complexes with FAD cofactor. Actually, these
xanthones showed interesting inhibitory activities towards AChE
and MAO, albeit no pronounced MAO isoform selectivity was
observed with the exception of 8a and 8b that were considerably
more active towards the A isoform. In the kinetic analysis,
xanthones 8a-c behave as mixed-type inhibitors of AChE and
compound 9 shows a non-competitive profile. These data suggested
a partial binding at PAS, and a possible consequent inhibition of -
amyloid formation induced by AChE.
MAO-Is with Additional Ion-Chelating and/or Antioxidant
Activities
The combination of different biochemical actions in a single
molecular entity sounds as a profitable approach to combat another
widespread ND with a multi-factorial nature, that is Parkinson’s
disease. As for AD, no therapy is currently available to stop or
reverse the neurodegenerative decline observed in PD patients.
Patients suffering PD manifest typical motor symptoms, including
postural instability, tremor, bradykinesia and rigidity [75]. These
clinical manifestations are the consequence of the CNS depletion of
dopamine, the neurotransmitter modulating the motor circuits in the
basal ganglia. Therefore, the mainstay of the current PD therapy is
based on the dopamine-replenishment strategy chiefly
accomplished with levodopa and/or dopamine agonists, and with
MAO-BIs as adjutants [76]. This therapy can be seen as a palliative
symptomatic treatment of motor deficits associated with PD as it
lacks the potential to alter the neurotoxic cascade. Beside the loss of
dopaminergic neurons mainly located in the substantia nigra pars
compacta, an additional histological hallmark in PD is the
accumulation of misfolded
-synuclein into intraneuronal fibrillar
aggregates, known as Lewy bodies (LBs) [77].
The prevention of
-synuclein-mediated neuronal toxicity has
been proposed as an alternative and valuable disease-modifying
approach in PD. Several biochemical events have been supposed to
promote
-synuclein fibrillogenesis, such as post-translational
modifications [78], oxidative stress [79], and the dyshomeostasis of
bioactive metal ions such as zinc, copper [80] and iron [81]. In
addition, the free iron ion seems to be one of the metals promoting
dopaminergic nigral neurodegeneration leading to PD [82]. On the
4576 Current Medicinal Chemistry, 2011 Vol. 18, No. 30 Pisani et al.

R1
R6 O OH
O OH
R5 R2
R4 O R3
O OH

(8a) (8b) (8c) (9)

R1 = R4 = OH; R1 = R6 = OMe; R1 = R5 = H;
R2 = R3 = R5 = H; R2 = R3 = R5 = H; R2 = R3 = R6 = OMe;
R6 = OMe R4 = OH R4 = OH
AChE Ki = 53.0 μM AChE Ki = 15.4 μM AChE Ki = 28.7 μM AChE Ki = 26.8 μM
MAO-A IC50 = 0.040 μM MAO-A IC50 = 29.0 μM MAO-A IC50 = 19.0 μM MAO-A IC50 = 37.4 μM
MAO-B IC50 = 33.0 μM MAO-B IC50 = 28% at 30 μM MAO-B IC50 = 14.7 μM MAO-B IC50 = 65.5 μM

Fig. (8). Natural non-alkaloidal dual MAO-AChE inhibitors 8-9 [74].

basis of such observations, iron chelation has the potential to
prevent ROS formation through Fenton reaction [83], the
consequent oxidative stress and the aggregation of
-synuclein [84].
Therefore metal chelation might represent a profitable approach to
efficiently target PD. Moreover, antioxidant compounds that are
able to reduce the rate of formation of toxic ROS might play a role
in PD.
Over the years the pivotal role of MAO-Is in PD therapy has
gained a growing consensus, since these drugs are able to limit
dopamine depletion. More recently, a strict connection of increased
MAO activity with high iron levels and oxidative stress conditions
has been postulated. Whether these mechanisms are really causative
or are manifestations of an ongoing neurotoxic cascade is still under
debate.
The multifaceted pathogenesis of PD prompted researchers to
develop therapeutic agents able to interfere simultaneously with
different targets in order to ameliorate the unsatisfactory current
pharmacological treatments. Indeed, a multipotent molecule with a
potential in PD can be obtained by the combination of appropriate
biological activities including an iron-chelating activity and a ROS
level reduction by radical scavenging mechanisms or throughout
MAO inhibition.
A multi-target ligand design approach has been applied by
Youdim and co-workers to obtain MAO-Is endowed with additional
iron-chelating and/or antioxidant properties (Fig. 9) by combining
the iron-chelating and antioxidant 8-hydroxyquinoline scaffold of
VK-28 (10) with a propargylamino group, which, along with MAO
inhibition, could offer an additional neuroprotective effect [85].
VK-28 has a potency for iron chelation equivalent to that of the
prototype iron chelator, desferal (Table 2) [85]. In contrast to
desferal, this iron-chelator was able to protect in vivo neurons from
toxicity deriving from 6-OHDA and MPTP neurotoxins in rats,
showing a favourable blood-brain barrier crossing capability [86].
Among these novel hybrid derivatives, two compounds, namely
HLA20 (11) and M30 (12) stand out for their highly relevant
biological activities [87]. In addition to their chelating power very
close to that of desferal, as measured in Fe2+-induced calcein
fluorescence quenching assays [85], both compounds inhibited
lipids peroxidation with IC50s in the low micromolar range (likely
through iron complexation) and showed higher MAO inhibitory
potency and brain permeability than VK28. The relative cell
permeability of 11 and 12 was measured through a chelator-induced
calcein fluorescence enhancement within the iron-quenched calcein
assay [85]. These multipotent ligands had a good permeability in
K562 cells that could be correlated to their favourable lipophilicity
as suggested by experimental logD determination. Further analyses
are needed to clarify their antioxidant profile that could be ascribed
to two possible mechanisms, that is the interference with Fenton
reaction and the radical scavenging properties. HLA20 (11) metal-

O O O O O
* CH3SO3H

H2N N N N N N
5 2 H 5 H 5
2
OH OH OH

Desferal

OH
N N
H
N N N N

N N N N
OH OH OH OH

VK28 (10) HLA20 (11) M30 (12) M30A (13)

Fig. (9). 8-Hydroxyquinolines 10-13 with MAO inhibitory, metal-chelating and/or antioxidant activities.
Targeting Monoamine Oxidases with Multipotent Ligands
Table 2. Biological Data of 8-Hydroxyquinoline Derivatives 10-13 [85, 87]
Enzymatic inhibitory activity
Compd
MAO-Aa (IC50) MAO-Ba (IC 50)
10 > 200 μM > 200 μM
11 300 μM 64.2 μM
12 0.037 μM 0.057 μM
13 2.39 μM 4.24 μM
desferal > 1000 μM > 1000 μM
a
Rat brain homogenates were used. Lipid peroxidation (LPO) was induced by [ascorbic acid] = 50 μM and [FeSO4] = 1.5 μM. [M]1/2 is the half maximal dequenching concentration
b c
of chelators in Fe2+-quenched calcein fluorescence test. Fe2+ binding affinity is inversely proportional to [M]1/2 value. dn.d. = not detected.
complexing property was more selective for iron than copper as
detected by spectrophotometric methods [85]. To investigate the
radical-trapping capability of 11, electron paramagnetic resonance
(EPR) spectra were registered with different concentrations of
scavenger in the presence of 5,5´-dimethyl-1-pyrroline-N-oxide
(DMPO) as a spin trap [87]. The concentration-dependent reduction
of the DMPO-·OH spin adduct signal in a H2O2 photolysis system
clearly indicated the ability of 11 to trap directly hydroxyl radicals.
Compounds M30 (12) and M30A (13) showed radical-
scavenging properties (data not shown) similar to 11 [87]. HLA20
(11) was a moderate MAO-I, less active but more MAO-B selective
than M30. On the other hand, both M30 and its demethylated
derivative M30A showed moderate selectivity for MAO-A in vitro
and in vivo. Their inhibition was time-dependent and of a suicide
type. Within this 8-hydroxyquinoline series, M30 is worth noting. It
proved to be a very potent albeit not selective MAO inhibitor with
potencies in the nanomolar range. Interestingly, M30 showed an in
vivo MAO inhibition profile similar to ladostigil (5b). M30 acts as a
brain permeable MAO-A and -B inhibitor without significant
effects on MAO activity in the liver and small intestine [88]. As a
consequence, M30 is able to enhance 5-HT and adrenaline neuronal
transmission, adding an antidepressant effect to its pharmacological
profile. The prominent, but still unclarified, central and unselective
MAO inhibition of M30 explains the lack of cardiovascular adverse
effects linked to tyramine potentiation.
Both M30 and M30A are metabolically transformed into active
propargylaminic metabolites that block the MAO catalytic activity
upon binding irreversibly and covalently to the prosthetic flavin
cofactor, FAD. The well-documented neuroprotective activity
associated to a propargylamino group, and unrelated to MAO
inhibition [89], claimed for a deeper exploitation of the potential
use of these multifunctional compounds to prevent neuronal cell
death on different in vitro and in vivo models. Concerning the
neuroprotective activity of HLA20, cell death induced by 6-OHDA
under oxidative stress conditions was studied in vitro using a PC12
cells model [87]. The neuroprotective power of HLA20 might be
ascribed to the combination of its multiple and different
biochemical properties, namely iron-chelation, MAO-B inhibition
and antioxidant activity. As for HLA20, a similar neuroprotective
ability against 6-OHDA toxic insults was observed for M30 [87]. In
a neurorescue trial performed on SH-SY5Y neuroblastoma cell
lines under serum deprivation conditions, M30 decreased apoptosis
likely through a reduced iron-promoted ROS production. In
addition, M30 is able to induce neuronal differentiation, blocking
cell cycle in G0/G1 phase [90]. Furthermore, in SH-SY5Y cells
M30 proved to reduce APP expression and shift APP processing
preferentially towards the non-amyloidogenic pathways [90]. Taken
together, these interesting features suggested M30 as a very
promising molecule for the treatment of NDs, such as PD and AD,
characterized by oxidative stress and iron dysregulation. In addition
to the neuroprotective and neurorescue effects mainly triggered by
the presence of the propargylamino moiety, the combination of
Current Medicinal Chemistry, 2011 Vol. 18, No. 30 4577
Antioxidant activity Chelating activity
LPO (Fe2+)a,b (IC 50) [M]1/2c
13.0 μM 4.80 μM
12.0 μM 5.40 μM
9.22 μM 6.50 μM
n.d. d
n.d.d
8.0 μM 1.50 μM
MAO-inhibitory activity with the iron chelating and antioxidant
properties by limiting two leading causes of ROS-related oxidative
stress might result in clinically relevant beneficial activities even
superior to rasagiline.
A further development of these 8-hydroxyquinoline-containing
metal chelators was focused on the improvement of their
pharmacokinetic properties, such as CNS permeability and on the
amelioration of their toxicity profile. Moreover a higher metal
selectivity was pursued to avoid an indiscriminate interference with
different metal ions pools, limiting their several important
physiological functions.
N
N
O O
N
(14)
AChE IC50 = 0.52
M
BChE IC50 = 44.9
M
MAO-A IC50 = 0.0077
M
MAO-B IC50 = 7.90
M
Fig. (10). Multifunctional pro-chelator and MAO-AChE inhibitor 14 [91].
A rational strategy to improve the drug-like properties of metal-
chelators is based on a prodrug or on a neutral amino acid
conjugation approach. To reduce citotoxicity and enhance
lipophilicity, a site activated pro-chelator of M30 was designed by
masking the 8-OH group of the quinoline moiety with a carbamate
group (Fig. 10) [91]. In addition, this structural modification holds a
potentially increased AChE targeting. The resulting compound (14)
was evaluated to determine its chelating properties and its ability to
inhibit both MAO and AChE in rat brain homogenates. As
expected, compound 14 per se was not able to chelate biometals but
was a potent and selective inhibitor of AChE (versus BChE) and
MAO-A (versus MAO-B). The central action of AChE, that is
located in different brain areas, allows to unmask the hydroxyl
group of 14 upon the carbamylation of the catalytic serine thus
releasing M30 and restoring the metal-chelating ability of the
molecule. UV-visible spectral bands shifts of 14 were observed
upon AChE-activation of the pro-chelator in the presence of metal
salts, thus proving its ability to chelate Fe, Cu and Zn ions. The
pronounced selectivity of this pro-chelator towards AChE may be
indicative of a safer profile, since it could reduce the toxicity
associated to the administration of non-selective AChE inhibitors as
tacrine, whose hepatotoxicity is attributed to some extent also to its
4578 Current Medicinal Chemistry, 2011 Vol. 18, No. 30
HO O
OH O
R = O-rhamnose Quercitrin
R = O-glucose Isoquercitrin
R = O-glucose-rhamnose Rutin
R = OH Quercetin
Fig. (11). Flavonoids 15 with dual MAO inhibitory-antioxidant activity [92].
low selectivity. In addition, the increased lipophilic character
allows the compound to cross the blood-brain barrier massively
thus obtaining a high CNS targeting while the low affinity for
BChE minimizes the peripheral cleavage and the unwanted
interference with metal homeostasis in peripheral tissues.
Compounds with Dual MAO Inhibitory-Antioxidant (Radical
Scavenging) Activity
As already discussed, one of the main factors triggering or
sustaining NDs may be the formation of free toxic radicals that
contributes to neuronal impairments by promoting abnormal
oxidized species in different biomacromolecules (e.g., lipids,
proteins and nucleic acids). The oxidative stress cascade can be
broken upstream, preventing the formation of hydroxyl radicals, or,
as an alternative, by sequestering intermediate radicals with
appropriate radical scavengers. Since a runaway free radical
production begins working in brain tissues suffering from
neurotoxicity and basal MAO catabolic activity contributes to ROS
production, the simultaneous inhibition of MAOs and the free
radical-scavenging activity through a single molecular entity could
potentially represent synergic activities that might result useful in
the treatment or prevention of NDs.
Flavonoids represent a class of compounds widely diffuse in
nature that are able to mediate different pharmacological actions,
often through antioxidant mechanisms. Four flavonoids, quercitrin
(15a), isoquercitrin (15b), rutin (15c) and quercetin (15d; Fig. 11),
have been isolated from the leaves of Melastoma candidum D. Don
and investigated for their ability to inhibit MAOs and to scavenge
free radicals [92]. The kinetic analysis of MAO-B inhibition
indicated a mixed-type mechanism, with Ki values in the low
micromolar range. Flavonoids 15 were endowed with hydroxyl
radical scavenging activity as assessed by an EPR spin trapping
technique. More recent results show that quercetin (15d) is a potent
and selective MAO-A inhibitor with an IC50 = 0.010 μM [93]. In
contrast with previous findings, it has been recently reported that
15d weakly inhibits MPTP bioactivation (33% at 10 μM
concentration) and this suggests also a limited inhibition of the
MAO-B activity [94].
O deamination reactions [100]. Thus the block of the production of
HO
Eugenol (16)
MAO-A Ki = 26.0 μM
MAO-B Ki = 211 μM
Fig. (12). Eugenol (16), a MAO inhibitor with antioxidant activity [96].
Eugenol (4-allyl-2-methoxyphenol, 16) is the major component
of clove, cinnamon, basil, and nutmeg oils and the major active
Pisani et al.
OH
OH
R
MAO-B Ki = 21.0 μM (15a)
MAO-B Ki = 2.72 μM (15b)
MAO-B Ki = 1.83 μM (15c)
MAO-B Ki = 7.95 μM (15d)
principle of Rhizoma acori graminei (RAG), a botanical remedy
described in Chinese pharmacopoeias and used as a palliative
treatment of AD cognitive dysfunctions. This compound holds an
antioxidant activity that could be ascribed to the phenolic structure.
It has been reported that 16 inhibits Fe2+-ascorbic acid induced lipid
peroxidation in a dose dependent manner, producing a 76%
reduction at a concentration of 250 μM [95]. To clarify the
mechanisms driving the antioxidant properties of 16, EPR spin
trapping experiments were performed. Eugenol antioxidant activity
is linked to its ability of trapping directly some ROS, such as
superoxide and hydroxyl radicals, rather than by acting as a free
radical chain-reaction breaker. In addition, eugenol can reduce ROS
production and relieve oxidative stress conditions through another
mechanism, that is a moderate and competitive inhibition of MAO
catalytic activity. In fact, 16 inhibits preferentially human
recombinant MAO-A (Ki = 26.0 μM) over MAO-B (Ki = 211 μM)
[96]. This dual activity of eugenol may represent a valuable feature
in the treatment of NDs.
Compounds with Dual MAO-NOS Inhibitory Activity
Different studies highlighted the effect of oxidative stress in
promoting mitochondrial dysfunction, inflammation processes and
nitric oxide (NO) toxicity in PD. Nitric oxide is a cellular signalling
mediator involved in several physiological and pathological
conditions [97]. Among others, NO regulates smooth muscle
relaxation, platelet aggregation, penile erection, macrophages
activities, brain functionalities (e.g., learning and memory).
Notwithstanding, NO is a free radical species in nature and its
overproduction may lead to harmful oxidative damages. NO can act
either as a weak oxidant or as reductive species and can exist in
three redox forms, namely nitric oxide (NO), nitrosonium (NO+)
and nitroxyl anion (NO–) [98]. It has been hypothesized that NO
toxicity is exerted through the production of reactive nitrogen
species (RNS; e.g., ·NO2, N2O3, HNO2) and by the reaction with
superoxide to form peroxynitrite anion (ONOO
), that is a strong
oxidizing agent involved in protein oxidation under physiological
conditions (Scheme 2) [99].
RNS and NO represent potentially mutagenic species able to
react with nucleic acids bases through nitration, nitrosation and
nitric oxide in appropriate brain areas might be an important goal in
the search of new agents able to halt or reverse neurodegenerative
cascades. To this end, one feasible approach has been envisaged to
discovery molecules with RNS scavenging property. In addition,
attention has been focused also on the design of molecules able to
inhibit the enzyme responsible for NO endogenous production, that
is the nitric oxide synthase (NOS) [101]. This enzyme catalyzes the
endogenous NADPH-dependent transformation of L-arginine to
citrulline and exists in three different isoforms: neuronal NOS
(nNOS), endothelial NOS (eNOS) and cytokine-inducible NOS
(iNOS). Thus, the reduction of the harmful NO-related biochemical
Targeting Monoamine Oxidases with Multipotent Ligands
thiols
(RSH)
N2O3
O2
L-arginine NOS NO
O2
ONOO
citrulline
OH
HNO2
Scheme 2. Endogenous NO and RNS production.
effects along the MAO-B inhibition might represent an original way
to slow down the rate of neurodegeneration.
R2
O N R3
N
R1
PF9601N (17) FA72 (18)
R1 = R2 = H R1 = R2 = R3 = H
R3 = _CH2C CH
nNOS IC50 = 187 μM nNOS IC50 = 192 μM
iNOS IC50 = > 1000 μM iNOS IC50 = 274 μM
LA oxidation IC50 = 82.8 μM LA oxidation IC50 = 60.2 μM
(rat liver) MAO-A IC50 = 1250 nM MAO-A = n.d.
(rat liver) MAO-B IC50 = 22.0 nM MAO-B = n.d.
Fig. (13). Indolyl dual MAO-I and NOS-I PF9601N (17) and its metabolite
FA72 (18) [103, 106].
Indole has been shown as a relevant scaffold in the inhibition of
MAOs [102]. SARs of a series of substituted indol-alkylamines
[103, 106], indicated that the introduction of a benzyloxy group in
position 5 of the indole ring remarkably shifted the selectivity
towards the B isoform. The suicide-type inhibition mechanism of
compounds of general structure described in Fig. (13) is due to the
presence of the propargylamino group. Among these indole
derivatives PF9601N (17) demonstrated a neuroprotective effect in
different models of PD [104]. Its ability to prevent the induction of
mitochondrial permeability transition pore seems to be independent
from MAO inhibition [105]. Therefore, to gain a deeper
comprehension of the neuroprotective activity, a study of the
antioxidant features and of the effect on NO-system of PF9601N
and its cytochrome P450–dependent metabolite FA72 (18) was
undertaken [106]. The radical-scavenging activities were evaluated
by measuring the reduction of oxygen consumption in a
peroxynitrite anion-promoted lipid peroxidation system. Both 17
and 18 displayed similar antioxidant properties in linoleic acid (LA)
as well as in brain homogenates models, without showing chain-
reaction breaking properties. This effect might be ascribed to a
direct interaction with peroxynitrite anion more than a free radical
scavenging. As a matter of fact, UV-visible absorbance spectral
changes were observed upon incubation of both inhibitors with
ONOO
. In addition, PF9601N and its metabolite FA72 were
moderate inhibitors of neuronal NOS, with IC50 of 187 and 192
μM, respectively. Interestingly, FA72 inhibits also the inducible
NOS isoform (iNOS) but with a high IC50 (274 μM). The synergic
action stemming from the inhibition of MAO-B, the effect on NO
system, the observation that a CYP-dependent metabolism was
active in various brain areas and that the resulting metabolite
Current Medicinal Chemistry, 2011 Vol. 18, No. 30 4579
GSH
RSNO GSNO
RSH
H+ NO2
ONOOH OH +
showed enzymatic and antioxidant activities comparable to its
parent compound, suggested PF9601N as a promising
neuroprotective agent for the prevention and/or cure of PD.
O
N
N Cl
O N N
(19)
MAO-B Ki = 0.181
M
NOS IC50 = 1057
M
O
N
N Cl
O N NH2
(20)
MAO-B Ki = 0.348
M
NOS IC50 = 577
M
Fig. (14). Dual MAO-NOS inhibitors 19-20 [107].
The discovery of a dual-targeting drug able to inhibit both
MAO-B and NOS was pursued also by Malan and co-workers
[107]. They took inspiration from the MAO-B inhibitory potency
observed in many xanthine derivatives, above all (E)-8-(3-
chlorostyryl)-caffeine (CSC, Fig. 17). For the design of dual MAO-
NOS inhibitors (Fig. 14), they followed a ring enlargement
approach of the bicyclic xanthine ring of caffeine to a pteridine-2,4-
dione ring that, while maintaining the structural features for the
molecular recognition of the MAO active site, might have promoted
NOS affinity because it structurally resembles the essential
tetrahydrobiopterin (BH4) cofactor. A potent and reversible MAO-
B inhibitor was identified (19) bearing a 6-styryl-pteridine-2,4-
dione structural framework, that was endowed with a nanomolar
potency towards MAO-B as proved by in vitro assays using baboon
liver mitochondrial fractions and measuring spectrophotometrically
the oxidation rate of MMTP to MMDP+. Unfortunately, 19 showed
a quite low NOS affinity as assessed in rat brain homogenates
containing several enzymatic isoforms. The authors ascribed the
failure of their design to the unforeseen steric hindrance of the
styryl group that would have hampered the targeting at the BH4
binding site of NOS. The iminic synthetic precursor (20) of the
target pteridine-2,4-dione 19 appeared less active towards MAO-B
but proved more effective in the inhibition of NOS.
The potent non-aminoacidic neuronal NOS inhibitor (nNOS-I),
7-nitroindazole (21, 7-NI, Fig. 15) [108], showed neuroprotective
properties against MPTP-induced parkinsonism in animal models
[109].
4580 Current Medicinal Chemistry, 2011 Vol. 18, No. 30 Pisani et al.

the thiocarbamoyl moiety strongly increased both analgesic and

N anti-inflammatory activity. Within this series, compound 22 is
N worth noting because it combines moderate MAO-B potency (Ki =
H 33.7 μM) with promising analgesic and anti-inflammatory activities
N comparable to those of indomethacin. Its analgesic and anti-
O O inflammatory profile was assessed in the p-benzoquinone-induced
7-Nitroindazole (21) writhing in mice and in carrageenan-induced hind paw oedema
model. As for the former, the analgesic activity was experimentally
(human) MAO-B IC50 = > 50
M proved by a 60% reduction in the number of writhings in mice.
(rat) nNOS IC50 = 0.71
M MAO-I 22 reduced markedly hind paw oedema promoted by
carrageenan administration. As a difference with known NSAIDs
Fig. (15). 7-Nitroindazole (21, 7-NI): dual MAO-NOS inhibitor [108, 109]. that show ulcerogenesis as a major side-effect, this derivative
displayed a promising low ulcerogenic liability.
To gain deeper insights into the biochemical mechanisms R2
underlying this effect, 7-NI was evaluated for its ability to interfere
with different steps of the biochemical cascade leading to MPTP
neurotoxicity. The neuroprotective action of 21 might be due to
some extent also to the reduced bioactivation of MPTP to MPP+ as
a result of a weak and competitive MAO-B inhibition [110]. N
N N
However, the main contribution of 7-NI to neuroprotection can H
arise from its antioxidant activity exerted through a powerful NH
hydroxyl radical scavenging property besides NOS inhibition [111]. S
Similarly, regioisomeric 5- and 6-nitroindazole can be considered R1
promising neuroprotective agents since they showed greater human (22)
MAO-B inhibitory potencies (IC50 = 2.5 μM and 6.8 μM,
R1 = Allyl; R2 = OMe
respectively) [109] along with a similar ·OH radical trapping ability
and a weaker in vitro inhibition of rat cerebellar NOS compared to MAO-B Ki = 33.7
M
7-NI (IC50 = 47.3 μM and 31.6 μM for 5- and 6-nitroindazole,
Fig. (16). Thiocarbamoyl-3-phenyl-5-(pyrrol-2-yl)-4,5-dihydro-(1H)-
respectively) [112]. Both these regioisomers were able to inhibit
pyrazole 22 with dual inhibitory and anti-inflammatory activity [117].
reversibly and competitively MAO-B mediated production of MPP +
with higher potencies than 7-NI (IC50 values equal to 0.99 μM, 2.52
μM, and 27.8 μM for 5-, 6-, and 7-nitroindazole, respectively) Compounds with Dual MAO-I and Adenosine A2A Receptor
[109]. Antagonist Activity
Currently, the main therapeutic strategy to cope with neuronal
Compounds with Dual MAO Inhibitory and Anti-Inflammatory dopamine deficit in patients suffering PD consists of a dopamine-
Activity replacement approach administering the direct biochemical
In view of the growing number of patients affected by AD, precursor of the neurotransmitter, namely levodopa (Chart 3), or
nowadays many novel molecular weapons are under investigation using receptor agonists that mimic the action of dopamine (e.g.,
for a neuroprotective or neurorestorative application, e.g., pramipexole and ropinirole, Chart 3) [76].
antioxidants, anti-amyloid agents, vitamins, estrogens, antagonists
N
of APO E4, growth factors, selective phosphodiesterase (PDE4)
O
inhibitors, and agents targeting neurotransmitters or neuropeptides
alterations [113, 114]. One of the most recently explored research HO
line involves the use of non-steroidal anti-inflammatory drugs OH
(NSAIDs), that in murine models of AD showed an interesting NH2
O
efficacy in the prevention of -amyloid deposition [115]. Though HO
their usefulness in a long-term administration regimen against AD N
H
is still a matter of debate because of possible side-effects [116], a
Levodopa Ropinirole
recent multi-target approach to obtain novel anti-AD agents is
focused on the synthesis and biological evaluation of compounds
with a dual MAO inhibitory and anti-inflammatory activity. N
Since pyrazole is the structural core of different NSAIDs, e.g. H2N
NH
celecoxib (a COX-2 inhibitor), and the same heterocyclic scaffold S
was present in several classes of MAO-Is, a series of 1-N-
Pramipexole
substituted thiocarbamoyl-3-phenyl-5-(pyrrol-2-yl)-4,5-dihydro-
(1H)-pyrazole derivatives were properly designed and evaluated, as Chart 3. Dopamine bio-precursor and agonists used in PD.
racemic mixtures, for their ability to inhibit MAO in rat liver
homogenates and exert an anti-inflammatory action (Fig. 16) [117]. Such a therapeutic approach offers a symptomatic relief of the
The analysis of SARs highlighted the key role of the substituent in characteristic motor deficits of PD but it is not able to target the
the para position of the phenyl ring. The compounds bearing a p- mechanisms underlying the dopaminergic dysfunction. Moreover,
chloro substituent were selective MAO-AIs with Kis in the range as the disease advances, progressive neuronal loss is observed and
13
61 μM. By replacing the chlorine atom in para position with an these drugs become ineffective. The inadequacy of dopamine-
electron-releasing group such as methoxy, a series of derivatives replacement therapies to perform a disease modifying action and
that selectively inhibit MAO-B isoform with Kis in the range 13
37 the side-effects of long-term use (e.g. choreiform, dystonic and
μM were obtained. The inhibition profile of all these compounds dyskinetic motor fluctuations) [118] prompted researchers to look
indicated a non-selective, irreversible and time-dependent for alternative strategies to find new, and druggable, biological
mechanism of inhibition. The presence of ethyl or allyl groups on
Targeting Monoamine Oxidases with Multipotent Ligands
Cl
O
N
N N
(E)
N (E)
O N
CSC (23)
A2A antagonism Ki = 30.2 nM
MAO-B Ki = 84.3 nM
Fig. (17). Caffeine derivatives 23-24 with dual MAO-I and adenosine A2A receptor antagonist activity [126].
targets that could be modulated in order to prevent, retard or halt
the progression of the neurodegenerative cascade leading to PD.
Antagonists of adenosine receptors that are selective for the A2A
subtype displayed a neuroprotective action [119] and were able to
prevent the onset of dyskinesia associated with long-term levodopa
treatment [120]. Thereby, their effects are additive to those of
dopamine-replacement therapy and may allow a reduction of
levodopa dosage regimen thus limiting the occurrence of adverse
effects. For these reasons, A2A antagonists emerged as a new class
of compounds with a potential value for neuroprotection and
symptomatic relief of motor deficits associated with PD [121]. As
previously mentioned, since MAO-B is the prevalent isoform
located in human basal ganglia and evidence of its, age-related,
increase has been reported by many authors [122], also MAO-B
inhibitors were considered a therapeutic opportunity and indeed are
used as adjutants of levodopa in the replenishment of dopamine
levels.
On this basis, research efforts have been directed to the
discovery of novel molecular entities with a dual action, that is
MAO-B inhibition and A2A receptor antagonism, that holds a
potential in the treatment of PD. Since experimental evidence
showed that caffeine, a non-selective A1/A2A receptor antagonist, is
able to protect from MPTP-induced neurotoxicity in mouse model
of PD [111], closely related heterocyclic derivatives were evaluated
too. Among them, (E)-8-(3-chlorostyryl)-caffeine (23, CSC, Fig.
17) [123] emerged as a potent and selective A2A antagonist
endowed with neuroprotective properties, that could be related to its
high affinity for MAO-B. Interestingly, CSC effect on the inhibition
of MAO-A was not significant [124]. Thus, different compounds
bearing a (E)-8-styrylxanthine scaffold were synthesized and
evaluated for a dual MAO-B/A2A targeting activity [125]. From the
analysis of SARs, it emerged that both biological actions were
favourably influenced by the introduction of a methyl group on the
xanthine N-7. The presence of an electron-donating group on the
styryl moiety strongly enhances MAO-B affinity, while reducing
A2A receptor antagonism not so dramatically. Since these novel
compounds proved to be from moderate to good MAO-B inhibitors
and A2A receptor antagonists, they deserved further investigations
to gain deeper insights into the molecular determinants of their
dual-targeting activity. With this purpose, the molecular framework
of the lead compound (CSC) was properly modified paying
O O
N N
R O
(25a)
R = N-ethyl-N-methylamino
Fig. (18). 7-Carbamoyl-1,2,3,4-tetrahydroisoquinoline derivatives 25 with dual MAO-I and APP processing modulator activity [129].
Current Medicinal Chemistry, 2011 Vol. 18, No. 30 4581
Br
O
(E)
N
N
O N
(24)
A2A antagonism Ki = 59.1 nM
MAO-B Ki = 21.9 nM
particular attention to the effects of the styryl group and its
substitution pattern [126]. Within this new series of compounds, the
phenyl and benzyl congeners resulted less active towards MAO-B.
The application of a vinilogous approach led to the (E,E)-8-(4-
phenylbutadien-1-yl)caffeine derivatives, among which 3’-
halosubstituted compounds showed outstanding MAO-B affinity
and potent A2A receptor antagonism, being both Kis in the
nanomolar range. The introduction of bromine in 3’-position led to
the most active dual-targeting derivative (24), that showed an
impressive high potency at both targets (Ki = 21.9 and 59.1 nM at
MAO-B and A2A, respectively). Further developments of these
compounds may be hampered by their likely low configurational
stability and high photosensitivity.
MAO-Is with APP Processing Modulatory Activity
The proteolytic cleavage of APP can be modulated at different
levels by intracellular second messengers. In this context, a pivotal
modulating role seems to be played by MAPK through the
activation of MEK/ERK signalling pathways [127]. It has been
reported that rasagiline (1) and ladostigil (5b) can stimulate the
release of the neuroprotective soluble form of amyloid precursor
protein
(sAPP
) by influencing the rate of PKC- and MEK-
related processing [128].
On the other hand, the reduction of toxic A1-42 production can
be achieved in a straightforward manner, through
- and -secretase
inhibitors, or indirectly, through the activation of PKC- and MEK-
dependent cleavage. The combination in one compound of some of
these biological activities, along with MAO-related ROS reduction,
may synergistically favour the APP non-amyloidogenic proteolysis,
or block the amyloidogenic one, thus deserving great attention for
the prevention and treatment of AD.
To achieve this goal, two series of 7-carbamoyl-1,2,3,4-
tetrahydroisoquinolines (25) were designed and synthesized as
cyclic congeners of ladostigil (Fig. 18) [129]. These sets of
derivatives differ for the substituent at basic nitrogen, where the
propargyl group was replaced by a more sterically hindered and
lipophilic cyclopropylmethyl group. All the synthesized derivatives
were evaluated as MAO inhibitors showing moderate IC50 values
ranging from 11.6 to 21.5 μM by using rat brain homogenates
(Table 3). Since cyclopropyl derivatives 25b-c were the most active
R O
(25b) (25c)
R = 1-piperidinyl R = 1-morpholinyl
4582 Current Medicinal Chemistry, 2011 Vol. 18, No. 30
Table 3. Biological Data of 7-Carbamoyl-1,2,3,4-tetrahydroisoquinoline Derivatives 25 [129]
Compd MAO-Ba inhibition (IC50)
25a 21.5 μM
25b 11.6 μM
25c 18.5 μM
a
Rat brain homogenates were used. Percentage of inhibition in the presence of 10 μM inhibitor. Activated (phosphorylated)/not activated ERK ratio in the presence of 10 μM
b c
inhibitor. dn.d. = not detected.
compounds for the inhibition of
-secretase enzymatic activity,
some of these compounds were tested in
-cells to detect their
influence on ERK signalling pathways. Interestingly, the most
potent MAO-B inhibitor (31b) was also able to inhibit
-secretase
and activate ERK pathways more strongly than rasagiline, showing
promising potential against AD [129].
Repositioning of Known Drugs as Multitarget Ligands with
MAO Inhibitory Activity
A recent research strategy in drug discovery, named “drug
repositioning” or “drug repurposing” and focused on the discovery
of new drugs for the therapy of neglected or orphan diseases,
consists in the analysis of approved drugs in the search of novel and
“hidden” pharmacological activities [4b]. Following this approach,
two known drugs have been proved to act as multi-target ligands
with interesting MAO inhibitory activities and a potential in NDs:
the antiepileptic drug zonisamide and the antidiabetic agent
pioglitazone (Fig. 19).
Zonisamide (26) is an FDA-approved drug for the treatment of
epilepsy that is also being used as an adjuvant in the therapy of PD
[130]. Its antiepileptic action is exerted through the blockade of
voltage-dependent Na+ channels and T-type Ca2+ channels [131]. It
has been reported that zonisamide is able to protect nigrostriatal
neurons from neurotoxic 6-OHDA insults [132], likely through a
radical-scavenging mechanism [133]. Its ability to reduce oxidative
stress prompted researchers to examine the effects of zonisamide in
animal models of PD and to define its mechanisms of action [134].
Zonisamide showed neuroprotection against MPTP toxicity in male
Swiss Webster mice and moderately inhibited mouse brain
homogenates MAO-B activity in vitro with an IC50 = 25 μM and a
reversible mechanism of action as assessed by ex vivo studies in
mice [134]. No inhibition of MAO-A activity was observed. More
recently, zonisamide complex with human MAO-B has been
solved, thus allowing a deeper comprehension of the binding
interactions at the active site of the enzyme [135]. Taken together
these findings underline the multi-target activity of zonisamide and
its potential use in PD, for a symptomatic improvement of motor
deficits and a neuroprotective action.
The peroxisome proliferator-activated receptor-
(PPAR
)
agonist pioglitazone (27) is a brain-permeable antidiabetic drug
belonging to the thiazolidinedione family. It has been demonstrated
that it performs a neuroprotective effect in animal models of AD
O
S NH2
O O
HN
N O
O
Zonisamide (26)
MAO-B IC50 = 25 μM
Fig. (19). “Repurposed drugs” inhibiting MAO-B with a potential in NDs [134, 139].
Pisani et al.

-secretase inhibitionb ERK activationc
26% n.d.d
72% 155%
62% 138%
showing an anti-inflammatory action [136]. In addition,
pretreatment of mice with pioglitazone reduced MPTP toxicity
[137] and dopaminergic neuronal loss [138]. The protective action
against MPTP toxicity is mediated by the inhibition of MAO-B
[139]. Pioglitazone showed a potent affinity in vitro towards human
recombinant MAO-B (IC50 = 220 nM) and its inhibitory efficacy
was proved also in ex vivo studies on male C57BL6/J mice. The
synergy of MAO-B inhibitory property and the ability to modulate
inflammatory responses in neurodegenerative processes through the
activation of PPAR
[140] increases the interest of this compound
as a potential therapeutic agent in NDs.
CONCLUSIONS
The rational design, synthesis and bio-pharmacological
evaluation of multipotent ligands addressing MAO inhibition
among other targeted pharmacological activities in NDs has been a
challenging research activity that led to the discovery of new
interesting bioactive compounds that deserve further development
towards pre-clinical and clinical trials. Although strong efforts have
been produced by several research groups in the rational molecular
design and in the explorative screening of large molecular libraries
to discover new promising multi-targeting agents for NDs, few
works succeeded in finding compounds with comparable potencies
or adequate potency ratio towards the addressed targets. These
difficulties arose mainly from the complexity of modulating
appropriately and in a simultaneous way two or more different
targets with a single molecule.
Unfortunately, the structure-based design of a multipotent
ligand is possible only in limited cases, since the 3D structures of
the targeted biomolecules are not always available. The lack of
information about the binding mode of a multipotent ligand into the
active sites of the diverse biological targets may force the
researcher to a sort of “blind design” characterized by a long
sequence of trials and errors. In these cases, indirect ligand-design
methods may be profitably applied through the derivation of
QSAR, [141] 3D-QSAR [142] and pharmacophore models [143]
that may allow a more rational and successful design of a
multipotent ligand. A more detailed knowledge of the main binding
interactions occurring with the different biological targets is instead
possible through structure-based computational methods which may
lead to an unequivocal identification of the molecular determinants
responsible for the activity at each single target and an easier
development of a multi-target pharmacophore model.
S
O N
Pioglitazone (27)
MAO-B IC50 = 220 nM
Targeting Monoamine Oxidases with Multipotent Ligands Current Medicinal Chemistry, 2011 Vol. 18, No. 30 4583

In some examples reported herein, additional activities DA = Dopamine

combined with a principal one, may be considered more properly DNA = Deoxyribonucleic acid
side-actions or additional effects that require a too high dose to
achieve the therapeutic threshold, thus increasing the risks of severe DMPO = 5,5´-Dimethyl-1-pyrroline-N-oxide
toxic side-effects. For the sake of completeness, we decided to 3D-QSAR = Three-dimensional quantitative structure-activity
report also those projects that surely need a further and sometime relationship
long optimization process to obtain a truly promising lead
compound. Taking into consideration the elegance of the design EPR = Electron paramagnetic resonance
strategy and the biological potential of these ligands, we reasoned eNOS = Endothelial NOS
that all the reported projects were worth of mention representing ERK = Extracellular-signal-regulated kinase
pioneering works in a fast developing field that may lay the
groundwork for future investigations. FAD = Flavin adenine dinucleotide
Regarding the multi-target ligands examined in the present FDA = Food and drug administration
review, a few compounds have been already submitted to deeper GRD = Glutathione reductase
pharmacological profiling and showed promising features but many
others need further and extensive development to improve their GSH = Glutathione
biopharmacological profile prior to any clinical trial. Above all, the GPX = Glutathione peroxidase
main limitation of the drug candidates emerging from a multi-target HD = Huntington’s disease
ligand design approach examined herein may mainly derive from
the lack of knowledge about the optimal balance among different 5-HT = Serotonin
pharmacological activities that could promote the desired iNOS = Inducible NOS
therapeutic effect and limit eventual side effects. This important
pharmaco-therapeutic aspect requires parallel pre-clinical studies on LA = Linoleic acid
structurally close active molecules showing different potencies and LBs = Lewy bodies
potency ratios of the targeted biopharmacological activities in vitro. LPO = Lipid peroxidation
This comparative analysis could allow the final selection of the best
clinical candidate exhibiting the most appropriate multi-targeting MAO-I = MAO inhibitor
profile, with ideal mechanisms of action, potencies and potency MAO-AI = Monoamine oxidase A inhibitor
ratio versus the different targets. It is worth noting that these pre-
clinical studies are much less demanding than those necessary to MAO-BI = Monoamine oxidase B inhibitor
develop any single component of a drug cocktail for NDs. In fact, MAP = Mitogen-activated protein
once the selection of the most promising candidate has been made, MAPK = Mitogen-activated protein kinase
the clinical study will be limited only to one compound. This
opportunity represents an undoubted advantage of a MTDL MEK = Mitogen-activated protein kinase kinase
approach compared to a polypharmacology strategy. MMTP = 1-Methyl-4-(1-methylpyrrol-2-yl)-1,2,3,6-
tetrahydropyridine
ACKNOWLEDGEMENTS MMDP+ = 1-Methyl-4-(1-methylpyrrol-2-yl)-2,3-
The financial support by MIUR-Rome (PRIN project dihydropiridinium
20085HR5JK) is gratefully acknowledged. MPP+ = 1-Methyl-4-phenylpyridinium
MPTP = 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine
ABBREVIATIONS
MTDL = Multi-target directed ligand
A
=
-Amyloid NA = Noradrenaline
AChE = Acetylcholinesterase ND = Neurodegenerative disorder/disease
AChE-I = Acetylcholinesterase inhibitor NFT = Neurofibrillary tangles
AD = Alzheimer’s disease 7-NI = 7-Nitroindazole
ADME = Adsorption distribution metabolism excretion NMDA = N-Methyl-D-aspartate
ALS = Amyotrophic lateral sclerosis nNOS = Neuronal NOS
APO E4 = Apolipoprotein E4 NO = Nitric oxide
APP = Amyloid precursor protein NOS = Nitric oxide synthase
Bad = Bcl-2-associated death promoter NOS-I = Nitric oxide synthase inhibitor
Bax = Bcl-2–associated X protein NSAID = Non-steroidal anti-inflammatory drug
BChE = Butyrylcholinesterase 6-OHDA = 6-Hydroxydopamine
Bcl-2 = B-cell lymphoma 2 PAS = Peripheral anionic subsite
Bcl-xl = B-cell lymphoma-extra large PD = Parkinson’s disease
BH4 = Tetrahydrobiopterin PEA =
-Phenethylamine
CAT = Catalase QSAR = Quantitative structure-activity relationship
CYP = Cytochrome P450 RAG = Rhizoma acori graminei
COX-2 = Cyclooxygenase-2 RIMA = Reversible MAO-A inhibitors
CSC = (E)-8-(3-Chlorostyryl)-caffeine RNS = Reactive nitrogen species
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= Protein kinase C
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