Alkaloids As A Source of Potential Anticholinesterase

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Review
Journal of Pharmacy
And Pharmacology
Alkaloids as a source of potential anticholinesterase

inhibitors for the treatment of Alzheimers disease
Eduardo Luis Konrath, Carolina dos Santos Passos, Luiz Carlos Klein-Jnior and Amlia T. Henriques
Programa de Ps-Graduao em Cincias Farmacuticas, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
Keywords
acetylcholinesterase inhibition; alkaloids;
Alzheimers disease; butyrylcholinesterase
inhibition; plants
Correspondence
Eduardo L. Konrath, Programa de Ps
Graduao em Cincias Farmacuticas,
Universidade Federal do Rio Grande do Sul,
Av. Ipiranga 2752, 90620-000 Porto Alegre
RS, Brazil.
E-mail: edukonrath@yahoo.com.br
Received February 28, 2013
Accepted May 12, 2013
doi: 10.1111/jphp.12090
Abstract
Objectives The inhibition of acetylcholinesterase (AChE), the key enzyme in the
breakdown of acetylcholine, is currently the main pharmacological strategy available for Alzheimers disease (AD). In this sense, many alkaloids isolated from
natural sources, such as physostigmine, have been long recognized as acetyl- and
butyrylcholinesterase (BChE) inhibitors. Since the approval of galantamine for the
treatment of AD patients, the search for new anticholinesterase alkaloids has escalated, leading to promising candidates such as huperzine A. This review aims to
summarize recent advances in current knowledge on alkaloids as AChE and BChE
inhibitors, highlighting structureactivity relationship (SAR) and docking studies.
Key findings Natural alkaloids belonging to the steroidal/triterpenoidal, quinolizidine, isoquinoline and indole classes, mainly distributed within Buxaceae,
Amaryllidaceae and Lycopodiaceae, are considered important sources of alkaloids
with anti-enzymatic properties. Investigations into the possible SARs for some
active compounds are based on molecular modelling studies, predicting the mode
of interaction of the molecules with amino acid residues in the active site of the
enzymes. Following this view, an increasing interest in achieving more potent and
effective analogues makes alkaloids good chemical templates for the development
of new cholinesterase inhibitors.
Summary The anticholinesterase activity of alkaloids, together with their structural diversity and physicochemical properties, makes them good candidate agents
for the treatment of AD.
Introduction
Alzheimers disease (AD) is a progressive age-related neurodegenerative disorder and is the most common form of
dementia among the elderly, generally diagnosed in individuals over the age of 65 years.[13] Considering the accelerated ageing of human society and the increase in life
expectancy worldwide, AD rates are predicted to increase
enormously, especially in developing regions. About 4.6
million new cases are added every year throughout the
world, and the World Health Organization (WHO) estimates that by 2040, 71% of the 81.1 million dementia cases
will occur in developing countries.[4,5]
The main pathological hallmarks of AD are the accumulation of amyloid plaques, or senile plaques, containing
extracellular deposits of b-amyloid peptide (Ab) and the
presence of intraneuronal neurofibrillary tangles (NFTs),
which result from hyperphosphorylated t-protein, both
producing neuronal cell loss in the nucleus basalis of

Meynert and in the hippocampus.[6,7] The most common
initial symptom of AD is deterioration of recent memory
with preservation of long term memory, leading to a progressive cognitive dysfunction, the main behavioural characteristic of AD.[8]
Although the exact pathogenesis of AD still remains to be
fully elucidated, it is currently considered to be a multifactorial disease. As such, different causes have been recognized
to be implicated in the course of AD, providing several
pharmacological strategies with multiple possible targets, all
of them under investigation.[9] Three main approaches have
been taken. The first involves the re-establishment of neurotransmitters levels, with the inhibition of cholinesterases,
acetylcholinesterase (AChE) and butyrylcholinesterase
(BChE), and also monoamine oxidase (MAO) enzymes.
2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 17011725
1701
Anticholinesterase alkaloids for AD
Eduardo Luis Konrath et al.
The second concerns neuroprotection, wherein oxidative

stress is considered to be an early event in the pathological
cascade for the disease, thus suggesting the potential use of
antioxidants to limit the effects of free radicals on nerve
cells. The third approach deals with specific aspects related
to AD, including the decrease in the production or aggregation of Ab peptide, and inhibition of g- and b-secretase
enzymes, which play a critical role in the amyloidogenic
pathway, t-protein,[10,11] among others.
Though recent intensive efforts have been made to
understand the mechanism of neurodegeneration involved
in AD and to discover new drugs combating the symptoms,
at present there is a deficit in the number of efficient and
safe therapeutic agents to treat the disease. No new drugs
have been approved by the US Food and Drug Administration (FDA) since 2003, likely because the abnormal brain
deposits of Ab and t-proteins still cannot be considered
causes or by-products of the disease.[12] The few current
drugs available only address the symptoms of cognitive loss,
without delaying or modifying the disease progression. In
some cases, the medications only work for a limited time
and in some patients they do not offer relief at all. Another
point to consider is that there is no in-vivo model available
that is able to mimic all the cognitive, behavioural, biochemical and histopathological abnormalities observed in
the course of AD, making it difficult to investigate new beneficial agents.[13,14]
The underlying principle of this review is to afford an
outline on the current status of isolated alkaloids with anticholinesterase properties, the main strategy available for AD
treatment, summarizing the most recent work in this area.
Their potency of inhibition for both AChE and BChE
enzymes is also reviewed, with the description of SARs,
when available. This review covers the main alkaloid classes
containing active molecules, including steroidal and triterpenoidal, quinolizidine (focusing mainly the Lycopodium
alkaloids), isoquinoline, indole and miscellaneous alkaloids.
The neuropathology of AD
Histopathologically, AD is characterized by the presence of
extracellular deposits of Ab, known as amyloid or neuritic
plaques, and intracellular NFTs containing abnormally
hyperphosphorylated t-protein, both processes resulting in
marked atrophy of brain tissue.[15] The neuropathology of
the disease is complex, and much effort has been devoted to
elucidating the relationships among these main hallmarks
and the aetiology of AD.
The density of senile plaques in the cerebral cortex is significantly correlated with the degree of cognitive impairment observed in patients before death, although amyloid
deposits can also be found in healthy brain tissue.[13,16] The
hippocampus and the basal forebrain neurons are damaged
1702
early in the course of AD, especially affecting the cholinergic

neurons and also causing pronouncing alterations in other
neurotransmitter systems, such as serotonin and glutamate.
Interestingly, a potential link between the two pathological
hallmarks of AD, senile plaques and NFTs, was also
described, since Ab deposits may induce t-phosphorylation,
resulting in tangle formation and, finally, neuronal death.[17]
Moreover, overproduction of Ab is also able to decrease
t-protein solubility in some cell lines.[18]
The amyloid hypothesis

Senile plaques are a constant feature of AD and consist of a
central extracellular Ab peptide core surrounded by degenerative nerve endings.[19,20] Ab is a small peptide (3943
amino acids in length) generated from a much larger
protein, the amyloid precursor protein (APP), which is a
transmembrane glycoprotein, consisting of three major isoforms of about 700 amino acids with a large extracellular
region, and is widely expressed in the brain.[21]
The sequential cleavage of APP is a complex process,
divided into the a- and b-pathways and performed by three
types of enzymes called secretases.[22,23] The a-pathway
involves the sequential cleavage of APP by a-secretase and
g-secretase, producing soluble peptides of non-amyloid
nature, P3 and sAPP, the former playing an important
role in synaptic plasticity and protecting neurons against
excitotoxicity. However, the sequential proteolysis of APP
by b-secretase, followed by an additional cleavage by
g-secretase, generates the insoluble Ab, which is highly
fibrillogenic and readily able to be deposited in amyloid
plaques. Since amyloid fibrils are protease-resistant structures, their formation cannot be reversed once initiated and
they act as a nucleation site for further aggregation.[24] The
toxicity of Ab42, the main species formed through the
b-pathway, is due to its insoluble b-sheet conformation
preferentially formed instead of an a-helix.[25] Another
point to consider is that the expression and activity of the
b-secretase enzyme is known to be increased with increasing age,[10] and this tends to contribute to the comparatively
high incidence of dementia in the elderly.
Investigations have suggested that overproduction of Ab
results in neurotoxic effects in neurons, leading to synaptic
dysfunction, formation of NFTs and, eventually, neuronal
loss.[26] However, it is unclear whether the amyloid plaques
are the causal agent or merely a by-product of neuronal
death. The amyloid cascade hypothesis considers that Ab is
the initiating molecule in the pathological process observed
in AD, and that it disrupts calcium homeostasis leading to
the generation of free radicals, lipid peroxidation and
reduced synaptic integrity. Moreover, small amounts of
soluble Ab have also been shown to compromise cholinergic functions, independent of cell death.[27] Therefore, parts
of the neurotransmitter deficit observed in AD can be

attributed to this phenomenon rather than to cell death.
Another effect resulting from amyloid deposition is the activation of glial cells and the expression of inflammatory
mediators in microglia and astrocytes, which leads to a scenario of chronic neuroinflammation.[23]
t-Protein and neurofibrillary tangles

NFTs are bundles of abnormal fibres located within
neurons and consist of pairs of filaments wound into paired
helical filaments. These filaments are made of t-protein,
which is a normal neuronal microtubule-bound protein
involved in the assembly and stabilization of microtubules.
In AD, t-protein becomes hyperphosphorylated as a result
of an imbalance between kinase and phosphatase activity,
which normally tightly regulate its phosphorylation. In this
condition, t-protein dissociates itself from the microtubules, loses its ability to stabilize them and self-aggregates,
forming insoluble oligomers and eventually microscopic
tangles.[28] However, these tangles are not specific to AD,
also being found in other neurodegenerative diseases; also,
infrequent cases of tangle-poor AD have been described.[29]
Cholinergic hypothesis
There is considerable evidence to suggest a possible association between the memory deficits described in AD patients
and impairment of cholinergic neurotransmission in the
central nervous system (CNS), in an attempt to characterize the cognitive and behavioural abnormalities
observed.[8,26,30,31] The cholinergic system plays an essential
role in the processes of learning and memory, and the dysfunction of this whole process is consistent with the progressive severity of memory impairment observed in AD.[32]
Hence, the cholinergic hypothesis postulates that restoration of cholinergic neurotransmission is a helpful strategy
to enhance the availability of synaptic acetylcholine and
ameliorate impaired memory in AD patients.[33,34] The conceptualization of AD as a cholinergic deficiency syndrome
provides a useful outline to the search for new drugs but it
is also important to understand that cholinergic deficit is
not the only neurotransmitter dysfunction observed in AD,
since catecholaminergic, glutamatergic and serotonergic
systems are also involved.[35] However, the few medications
available, with the exception of the N-methyl d-aspartate
antagonist memantine, only address the symptoms of cognitive loss by targeting the AChE enzyme inhibition.
Acetylcholine released into the synaptic cleft is quickly
hydrolysed by AChE, and the blockade of this catabolic
process results in increased levels of the neurotransmitter,
which may partially correct the cholinergic deficiency seen
in AD. BChE, or pseudocholinesterase, plays only a minor
role in regulating the acetylcholine levels when compared
with AChE,[36] but it does plays an important role in the

pathology of AD, since both enzymes are present in the
brain and are detected among NFTs and amyloid plaques.[37]
Various studies have indeed supported the hypothesis that
cholinesterase inhibitors can prevent Ab oligomerization,
thus displaying both anti-amyloid and neuroprotective
disease-modifying effect.[38] Moreover, in AD patients, the
level of AChE activity tends to decline and the activity of
BChE increases, with the ratio between BChE and AChE
changing from 0.6 in the normal brain to 11 in cortical
areas affected by the disease. In late AD stage, BChE represents the predominant cholinesterase in the brain, and for
this reason this enzyme is also targeted as an approach to
intercede the disease.[39,40]
The use of cholinesterase inhibitors

The currently approved treatment approach for AD focuses
on replacement therapy for deficits in central cholinergic
neurotransmission, with encouraging results obtained with
the use of anticholinesterases to amplify the physiological
action of acetylcholine in AD patients.[38,41,42] Three drugs
that target the cholinergic system are currently approved by
the FDA for AD: donepezil, rivastigmine and galantamine
(Figures 1 and 2).
Tacrine was the first medication approved by the FDA
and introduced in the market for the treatment of AD in
N
O
O
O
Donepezil
NH2
N
N
O
O
N
Rivastigmine
OH
Tacrine
NH2
N
Velnacrine
Figure 1
disease.
Some cholinesterase inhibitors used to treat Alzheimers
1703
NH
2
10
10a
O
9
1
10b
D
6a
4
4a
11
OH
3
12
Galantamine
Physostigmine
H
N
NH2
Huperzine A
Figure 2 Some natural alkaloids used as cholinesterase inhibitors for
AD therapy.
1993, providing cognitive improvement in 540% of

patients with mild-to-moderate AD after treatment.[43,44]
However, gastrointestinal side effects and serious doserelated hepatotoxicity were reported, frequently preventing
dose escalation to 160 mg, the dosage required for
maximum proven efficacy on cognition.[45] A major
metabolite of tacrine, velnacrine, was also screened as a
cholinesterase inhibitor, although the molecule also demonstrated significant hepatotoxicity in vivo.[46]
In 1996, donepezil was cleared by the FDA for AD, and
was considered to have better properties than tacrine, with
lower toxicity and higher selectivity for AChE, and was also
able to increase the density of nicotinic receptors in the
brain. This piperidine derivative has a longer half-life than
tacrine, but has a more moderate efficacy than that produced by the maximum recommended dose of tacrine.
However, donepezil does not produce hepatotoxicity, and
has few side effects, all of them related to gastrointestinal
system.[47,48] Thereafter, the use of rivastigmine was
approved in 2000, being first marketed in Switzerland for
the treatment of AD. This physostigmine derivative, which
is administered twice daily, inhibits both cholinesterases
with a similar affinity, and it does not cause liver damage,
producing only cholinergic side effects such as nausea
and vomiting.[46,49] Galantamine, unlike the other anticholinesterases, is an alkaloid obtained from natural
sources, first isolated from Galanthus nivalis L and related
species belonging to the Amaryllidaceae family. This
product was first marketed in 2001 and is a long-acting,
relatively selective AChE inhibitor, with less BChE inhibitory activity and some gastrointestinal side effects
1704
described.[50,51] Moreover, galantamine also improves

cholinergic transmission by allosteric modulation of nicotinic receptors and has been used to treat the symptoms of
other forms of dementia.[52] It is currently isolated from
daffodil bulbs by the pharmaceutical industry, which has
engendered an exponential interest in the isolation and
characterization of alkaloids from Amaryllidaceae bulbs
(e.g. lycorine, homolycorine and hipeastrine), most of them
having anticholinesterase activity.[53]
The use of cholinesterase inhibitors appears to have beneficial impact on both behavioural and psychiatric symptoms, but there is no evidence for superiority of one agent
over another with respect to cognitive, behavioural or functional outcomes.[10,54] Several agents targeting AD are currently under clinical trials but failure rates are high, which is
an unfortunately common point for drugs that target the
brain. In this sense, researchers are currently focusing on
biomarkers to provide insight into disease progression,
hoping to find therapies that can be applied at parts of the
disease mechanism that occur at an early stage in the
brain.[12]
Alkaloids as anticholinesterase
inhibitors
A number of AChE and BChE inhibitors have been isolated
from various natural sources, as extensively described in the
literature.[5560] It is noteworthy that most of the compounds
have demonstrated only in-vitro activity; few of them have
been tested on animal models, which is imperative to prove
their capacity to cross the bloodbrain barrier and exert
beneficial effects in the brain.
Among these natural products, alkaloids are considered
to be the most promising candidates for use in the treatment of AD, due to their complex nitrogen-containing
structures.[61] In fact, one of the binding sites of AChE
involves the interaction of the positively-charged nitrogen,
even though other binding site exists, in order to allow the
inhibition by non-alkaloid compounds, mainly terpenes,
xanthones and coumarins.[56,57,62]
AChE inhibitors have been extensively studied as therapeutic options for AD after the initial observation that
physostigmine (eserine), an alkaloid obtained from the
seeds of Physostigma venenosum Balf. (Fabaceae), traditionally used as a ritual poison in Africa,[63] could reverse the
disruption of cognition produced by scopolamine in animal
models. This alkaloid has a rapid effect and although it acts
as a reversible inhibitor for both cholinesterases, it is more
selective for AChE. However, the use of physostigmine is
currently limited by its short half-life, its narrow therapeutic window and its gastrointestinal and orthostatic
effects.[64,65] Finally, molecular modifications of the alkaloidal structure of physostigmine have led to the development
of its analogue rivastigmine, a synthetic compound with a

better therapeutic and safety profile.
Other anticholinesterase alkaloids currently studied for
their use in AD include galantamine, as previously reported,
and huperzine A, a quinolizidine-type alkaloid isolated
from Huperzia serrata (Thunb) Trev, a Chinese species
traditionally used for the treatment of swelling, strains,
schizophrenia, myasthenia gravis and organophosphate poisoning.[66,67] Huperzine A has been found to be a potent,
reversible and selective AChE inhibitor, with oral bioavailability, able to cross the bloodbrain barrier and act with a
prolonged half-life.[68] Investigations conducted with this
alkaloid demonstrated it to exert a more potent effect in
increasing the cortical acetylcholine levels when compared
with either donepezil or rivastigmine and it also demonstrated higher in-vivo AChE inhibition.[69] Clinical trials at
phase IV, conducted in China, showed that huperzine A significantly improved memory shortages in elderly people
with benign senescent forgetfulness and in patients with
AD, with minimal peripheral cholinergic side effects and
toxicity.[70,71] However, recent phase II clinical trials conducted in the USA did not demonstrate any beneficial
cognitive effect of huperzine A in patients with mild-tomoderate AD, suggesting that further investigations must be
conducted to prove the efficacy of this compound.[72] In
this sense, Lycopodium alkaloids have become a focus of
research in recent years, resulting in many in-vitro and
in-vivo pharmacological studies aimed at cholinesterase
activity. However, all of the other Lycopodium alkaloids
identified so far have either shown no cholinesterase inhibitory activity or have possessed in-vitro activity significantly
lower than that of huperzine A.[73]
Many alkaloids that are active cholinesterase inhibitors
have already been described in different families, including
the isoquinoline-type alkaloids from Amaryllidaceae (especially found in Narcissus sp, Galanthus sp, Hyppeastrum
sp)[74,75] and Papaveraceae,[76,77] the steroidal alkaloids
from Buxaceae (genera Buxus and Sarcococca),[78] the
quinolizidine-type alkaloids from Lycopodiaceae (genera
Huperzia and Lycopodium)[7981] and the indole alkaloids
from Apocynaceae and Rubiaceae[82] among others.
Although the great interest in these products has inspired
developments in medicinal chemistry, leading to the synthesis of more potent synthetic analogues, reviews focusing
only on anticholinesterase alkaloids obtained from natural
sources are scarce in the literature.
Classes of anticholinesterase
alkaloids
Steroidal and triterpenoidal alkaloids
The active alkaloids belonging to this class are distributed in
species of Solanum, Veratrum and Fritillaria and in the Bux-
aceae family, mainly in Buxus and Sarcococca genera, as

shown in Figure 3.[83] The Sarcococca genus is known for the
production of pregnane-type steroidal alkaloids. These
metabolites have mono- or dimethylamino substituents at
the C-3 and/or C-20 position in the basic steroidal skeleton.[84] Several Sarcococca steroidal alkaloids have been
reported to have anticholinesterase activity, with IC50
values in the range of 0.5-249 mm for AChE, (+)phulchowkiamide A being the most active, while for BChE
the IC50 varies from 0.3 to 200 mm, hookerianamide I possessing the lowest IC50 value (see Table S1).[8595] Through
LineweaverBurk and Dixon plots, Khalid et al.[84] demonstrated that the majority of the pregnane-type alkaloids
obtained from Sarcococca saligna have a pure noncompetitive type of inhibition for both cholinesterases.
Despite the similar activity range, this type of alkaloid is,
in general, more selective for BChE. The selectivity might be
explained by the larger size of the steroidal alkaloids, which
can easily diffuse into the larger aromatic gorge of BChE,
when compared with AChE. The residues of Phe-330 and
Tyr-121 in AChE form a bottleneck, making the access of
these compounds into the gorge difficult. Another important aspect that might also play a role in the selectivity of
these compounds is that hydrophobic interactions are more
prominent in BChE.[92] SAR studies demonstrated that the
amino nitrogens at C-3 and/or C-20 positions are the most
important features related to the potency of the compounds. At physiological pH, these nitrogens are expected
to be protonated and to have a similar behaviour to some
known quaternary and bisquaternary inhibitors.
Despite the higher selectivity for BChE, quantitative SAR
(QSAR) and docking studies of these Sarcococca alkaloids
focus on AChE. Through a 3D-QSAR study by comparative
molecular field analysis of 32 steroidal alkaloids obtained
from S. saligna, Zaheer-ul et al.[96] inferred that the presence
of negative density close to the substituent on the C-3
side chain and C-4 positions increase the AChE inhibitory
activity. On the other hand, negative density close to
C-2 decreases the inhibitory activity. For instance,
2b-hydroxyepipachysamine-D is almost three-fold less
active than epipachysamine-D, which does not have a
hydroxyl group at C-2. Furthermore, the presence of a
double bond between C-5 and C-6 also seems to increase
the activity, since saracocine, with a double bond in ring B,
is about two-fold more active than sarcodine, which does
not possess this unsaturation. Regarding the bulky groups,
their presence near the side chain at C-3, C-4, C-14 and
C-15 is not favourable for AChE inhibitory activity.
In a docking study using the crystal structure of Torpedo
californica AChE co-crystallized with decamethonium, a
series of pregnane-type steroidal alkaloids was evaluated,
demonstrating that all the 15 compounds adopted the same
binding mode. The ring A of the steroidal skeleton enters
1705
NH
NH
H
H
O
H
H
N
NH
H
O
(+)Phulchowkiamide A
Hookerianamide I
21
20
18
11
19
HO
10
2
3
NH
H
6
12
13
17
16
14 15
H
O
NH
2-Hydroxyepipachysamine-D
Epipachysamine-D
Ac
H
H
H
N
H
Sarcodine
Saracocine
N
O
H
OH
O
MeO
N
H
NH
H
HO
OAc
OMe
O2 -Natafuranamine
Figure 3
1706
Buxakashmiramine
Steroidal and triterpenoidal alkaloids with anticholinesterase activity isolated from natural sources.
NH
NH
H
Buxamine C
Buxamine B
21
20
21
O
12
11
9
1
NH
10
2
4
13
14
18
17
15 16
8
7
19
H
(+)Buxabenzamidienine
(+)Buxamidine
27
25
26
N
22
18
H
19
10
5
14
20
16
21
17
HO
12
13
15
1
2
3
11
9
24
23
HO
H
O
Chuanbeinone
Ebeiedinone
21
N
18
19
11
9
10
5
3
4
H
6
12
H
8
7
20
H
17
16
15
13
14
Conessimin
Figure 3
Continued.
1707
into the aromatic gorge and is placed into the bottom,

which might be due to the greater hydrophobicity of this
ring in comparison with the ring D. Also, the alkaloids are
buried inside the aromatic gorge, significantly contributing
to the stabilization of the complex, but these compounds
are not as deep into the aromatic gorge as the ligand.
Despite this, amino groups at C-3 and C-20 come close to
Tyr-331 and Phe-281, demonstrating a similar binding
position when superimposed with the co-crystal. Nevertheless, electrostatic interactions do not contribute significantly
to the binding energy, since the most important contributions are guaranteed by van der Waals interactions. The
binding of the alkaloid with the enzyme does not cause
major structural rearrangements, although the dynamics of
the gorge are considerably changed, since its flexibility is
reduced.[97]
Another well-studied genus from the Buxaceae family is
Buxus, considered to be a rich source of cycloartenol-type
triterpenoidal alkaloids. As in the pregnane-type alkaloids
obtained from Sarcococca, nitrogens atoms are also present
at C-3 or/and C-20,[98] and for this reason they are also recognized as a source of anti-AChE and -BChE compounds.
The IC50 values range from 3.0 (O2-natafuranamine) to
468 mm for AChE, while for BChE the values range from
0.74 (buxakashmiramine) to 350 mm.[99104] Also, Khalid
et al.[105] demonstrated that buxamine B and buxamine C
have a non-competitive inhibitory profile for AChE, as verified by LineweaverBurk plots.
Orhan et al.[106] reported the specificity of (+)buxabenzamidienine (10-fold) and (+)-buxamidine (more
than 320-fold) for AChE inhibition. However, Atta-urRahman et al.[101] and Choudhary et al.[100,102] observed that
usually the triterpenoidal alkaloids were more selective for
BChE. Some docking studies performed with Torpedo californica AChE and Buxus alkaloids demonstrated that, as
observed for the steroidal alkaloids, the C-3 and C-20
amino groups are essential for the activity, and that the
hydrophobicity of the compounds is also related to the
inhibitory capacity of the ligands.[105,106] Corroborating these
results, Choudhary et al.,[102] through SAR studies of nine
different triterpenoidal alkaloids, demonstrated that the
presence of Me2N moieties at C-3 and C-20 may be the
most important features for the anticholinesterase activity.
Moreover, the presence of substituents at C-16 and at C-31
decreases the inhibitory effect. On the other hand, unsaturated sites do not seem to influence the activity, since the
aromatic gorge of the enzymes are extremely flexible, facilitating the accommodation of different conformations of the
alkaloids.
Some Fritillaria species have also been studied due to the
anticholinesterase activity of their steroidal alkaloids.
Usually, these compounds possess a C27 cholestane carbon
skeleton, with five or six carbocyclic or heterocyclic
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rings.[107] As for the other steroidal alkaloids, the Fritillaria

alkaloids are more selective for BChE, with IC50 values
ranging from 0.7 to 121 mm, chuanbeinone being the most
active. For AChE, the IC50 varies from 5.7 (for ebeiedinone)
to 352 mm.[108,109] In a SAR analysis of a series of these steroidal alkaloids, it was observed that the potency is increased
by the presence of functional groups at C-3 and by the C-6keto function. In contrast, hydroxyl substitution at C-20
and methyl substitution at the N position are responsible
for decrease in the anticholinesterase activity.[108]
Yang et al.[110] isolated five different steroidal alkaloids,
sharing a fused-pyrrolidine ring at the preganane skeleton,
from the species Holarrhena antidysenterica and evaluated
their capacity to inhibit AChE, with IC50 ranging from 4 to
28 mm. The most active compound, conessimin, demonstrated a reversible and non-competitive mode of inhibition. The pyrrolidine moiety of the alkaloid forms a
hydrogen bound with Tyr-72. Through a SAR analysis of
the five alkaloids, it was observed that the N-Me substituent
of the pyrrolidine moiety seems to decrease the activity,
while the presence of the same substituents at C-3 increases
the anti-AChE effect.
Quinolizidine-type/Lycopodium alkaloids
Lycopodium alkaloids are quinolizine, pyridine or
a-pyridone alkaloids, which may be divided into four different classes: lycopodine, lycodine or flabellidine, fawcettimine and miscellaneous. The most prominent compound
belonging to this group, huperzine A, is a lycodine-type
alkaloid and was first isolated from the Chinese lycopod
Huperzia serrata (Thunb. Ex Murray); it is responsible for
the exponential increase in interest on investigations of
Lycopodiaceae species (Figure 4), as previously cited.[73]
Halldorsdottir et al.[111] isolated ten known lycopodane alkaloids from Lycopodium annotinum ssp. alpestre collected in
Iceland, and evaluated their in-vitro anticholinesterase
activity. As a result, the most potent inhibitors of AChE
were anhydrolycodoline (IC50 = 191 mm), and lycofoline
(IC50 = 600 mm), while for BChE only lycodoline could
inhibit the enzyme activity, with a very high IC50 value
(667 mm) (Table S2). In an attempt to understand the
reason behind the low anticholinesterase activity of the
lycopodane alkaloids (some of them have an IC50 value
higher than 2000 mm), investigations using docking studies
were made using Torpedo californica AChE co-crystallized
with huperzine B. Comparing the orientations assumed
by anhydrolycodoline and by huperzine B, the authors
observed that both were completely different, since the carbonyl groups of the two molecules were pointing in opposite directions. Also, the nitrogen of anhydrolycodoline was
shifted about 3.0 from the position of the nitrogens of
huperzine B, and no stabilizing hydrogen bonds were
OH
HO
OH
Anhydrolycodoline
Lycofolin
Lycodoline
HO
O
N
H
NH
H2N
N
H
Cryptadine B
Carinatumin A
OH
OH
O
O
H
H
NH
HN
NH
OH
O
Lycoparin A
Cernuine
Sieboldine A
HO
Lycoparin B
OH
Lycocernuine
H
H
O
Acetyldihydrolycopodine
Figure 4
Lycopodine
Quinolizidine/Lycopodium alkaloids with anticholinesterase activity isolated from natural sources.
1709
detected. Finally, the authors concluded that the lycopodane

alkaloids fit into the binding site of AChE, although they
are not able to form strong interactions with the amino
acids of this site, thus explaining their high inhibitory
concentrations.
Some miscellaneous Lycopodium alkaloids were also
evaluated due to their capacity to inhibit AChE. The most
active, cryptadine B (IC50 = 18.5 mm) isolated from L. cryptomerinum, consists of two octahydroquinoline rings and a
piperidine ring.[112] However, other alkaloids with the same
C27N3 pentacyclic skeleton inhibited the enzyme activity
with a higher IC50 value (>60 mm).[112,113] On the other
hand, lycodine alkaloids, in the class of huperzine A, as
expected were much more active (e.g. carinatumin A, isolated from L. carinatum, exhibited an IC50 value of
5.6 mm).[114] Even so, some other alkaloids of the same
class are considered inactive (IC50 > 200 mm), such as
lycoparins A and B.[115] Interestingly, one of the most
active Lycopodium alkaloids, with an IC50 value of 2.0 mm,
is sieboldine A, a fawcettimine-derived compound with
a fused tetracyclic ring system, consisting of an azacyclononane, a cyclohexanone, a cyclopentanone and a tetrahydrofuran ring.[116]
Recently, Konrath et al.[81] investigated some Lycopodiaceae species belonging to Huperzia and Lycopodiella
genera collected in Brazil, which resulted in interesting
profiles of anticholinesterase inhibition for the alkaloid
extracts. Some compounds isolated from these species were
also screened in vitro, but the results showed a lower
potency of inhibition when compared with the IC50 values
from the alkaloidal extracts, suggesting a synergism among
the compounds. For example, Lycopodiella cernua extract
showed an IC50 value of 42.6 mm, while its main alkaloids
cernuine and lycocernuine possessed IC50 values of 32.7 and
>250 mm, respectively. Moreover, acetyldihydrolycopodine,
one of the main alkaloids found in L. clavatum and L. thyoides collected in Brazil had an IC50 value of 84.7 mm, while
lycopodine had a much lower anticholinesterase activity,
suggesting that the acetyl moiety may have a beneficial
influence on enzyme inhibition.[117]
Isoquinoline-type alkaloids
Amaryllidaceae alkaloids
The Amaryllidaceae alkaloids represent a large group of isoquinoline alkaloids, the majority of which are known to
occur exclusively in this family. These alkaloids can be classified into nine skeleton types, for which the representative
ones are: norbelladine, lycorine, homolycorine, crinine, haemanthamine, narciclasine, tazettine, montanine and galantamine.[118] Regarding AChE inhibition, the most active
compounds, displaying IC50 values close to 10-6 m, belong to
the galantamine type (Table S3). In a study evaluating 23
1710
Amaryllidaceae alkaloids belonging to the lycorine, homolycorine, haemanthamine, galantamine, tazettine and miscellaneous types, Lopz et al.[74] demonstrated that sanguinine,
galantamine, 11a-hydroxygalantamine, and epinorgalantamine inhibited AChE from the electric eel, Electrophorus
electricus (EeAChE), with IC50 values varying from 0.10 to
9.60 mm. The authors suggest that the AChE inhibitory
effects seem to be related to structural characteristics
among the different skeleton types, as only the alkaloids
included in the galantamine and lycorine (assoanine,
oxoassoanine, pseudolycorine) groups exhibited such
inhibitory activity. The higher potency of hydroxygalantamine for AChE inhibition, in comparison with galantamine, may be due to the presence of an additional
hydroxyl group in sanguinine, which could aid in the stabilization of the ligand in the enzyme binding pocket by classical and non-classical hydrogen bonds.[74,119] Chlidanthine,
a positional isomer of galantamine, is about 10-fold less
potent for AChE inhibition (EeAChE IC50 = 24.1 mm).[120]
This difference in activity could be assigned to the stereochemistry of chlidanthine, which hinders the establishment of intramolecular hydrogen bonds between the
hydroxyl group at C-3 and the oxygen atom of the dihydrofuran ring, such as occurs in galantamine ring.[120,121]
Among the other subclasses of Amaryllidaceae alkaloids,
the most potent cholinesterase inhibitors seem to be those
compounds belonging to the lycorine type. Assoanine
(EeAChE IC50 = 3.87 mm), oxoassonine (EeAChE IC50 =
47.2 mm)[74] and 1-O-acetyllycorine (IC50 = 0.96 mm)[53] are
the most potent AChE inhibitors in this group, their
potency being attributed to (i) the presence of an aromatic
ring C, for assoanine and oxoassonine, and (ii) the acetoxy
and hydroxyl substituents attached to C-1 and C-2 of 1-Oacetyllycorine. Ungeremine, a quaternary lycorine-type
alkaloid with the ring C aromatic, inhibited EeAChE displaying an IC50 of 0.35 mm.[122] The higher potency of this
compound for EeAChE can be assigned to the presence of
the ring C aromatic together with the quaternary nitrogen
atom, which is important for the inhibitory activity displayed by other isoquinoline alkaloids, such as berberine.
Furthermore, the methyl and epoxy groups present in
incartine (EeAChE IC50 = 107 mm) and incartine N-oxide
(EeAChEIC50 = 34.5 mm) also favour AChE inhibition.[123]
Amaryllidaceae alkaloids possessing the other skeleton seem
to behave as weak cholinesterase inhibitors with IC50 values
close to 10-4 to 10-3 m.[53,124,125]
Other isoquinoline alkaloids
The activity of protoberberine alkaloids on cholinesterases
has been evaluated by numerous authors, mainly regarding
the quaternary compounds berberine and palmatine, as
seen in Figure 5. Ingkaninan et al.,[126] in a study with proto-
OH
OH
O
O
HO
OH
N
11-Hydroxygalantamine
Sanguinine
2
3
OH
O
10
O
9
11
10a
6a
7
NH
12
6
Assoanine
Epinorgalantamine
OH
HO
O
H
HO
N
O
H
N
Pseudolycorine
Oxoassoanine
OAc
OH
AcO
O
O
H
1-O-Acetyllycorine
Chlidanthine
Figure 5
Isoquinoline-type alkaloids with anticholinesterase activity isolated from natural sources.
berberine alkaloids from Stephania venosa, verified that

the quaternary compounds stepharanine (EeAChE IC50 =
14.1 mm), cyclanoline (EeAChE IC50 = 9.23 mm) and Nmethyl stepholidine (EeAChE IC50 = 31.3 mm) were about
five-fold more potent for EeAChE inhibition than the

tertiary compounds isolated from the same plant
(IC50 > 100 mm). Additionally, the effects of the alkaloids
from S. venosa on EeAChE were compared with those
1711
OH
N+
O
O
N+
O
Ungeremine
Berberine
O
HO
HO
H
N+
O
Incartine
Incartine N-oxide
O
+
N+
HO
O
O
OH
Stepharanine
Palmatine
OH
O
O
H
OH
H
HO
N+
O
Cyclanoline
Figure 5
1712
N+
HO
N-methyl stepholidine
Continued.
HO
O
O
Jatrorrhizine
O
Chelerythrine
HO
N
H
NO2
OH
HO
NO2
NO2
O
()2,9-Dihydroxyl-3,11-Dimethoxy1,10-Dinitrotetrahydroprotoberberine
(+)Nitroapocavidine
O
O
O
O
Sanguinarine
Dehydrocavidine
O
+
HO
Dehydrocorydaline
Figure 5
Pseudocolumbamine
Continued.
displayed by the well-known quaternary alkaloids berberine

(EeAChE IC50 = 0.58 mm), palmatine (EeAChE IC50 =
0.26 mm) and jatrorrhizine (IC50 = 0.93 mm). Besides
EeAChE, berberine and palmatine were also tested in
human, mice and rat enzymes displaying about the same
potency.[127132] Regarding BChE, both compounds inhibited

equine serum enzymes, displaying IC50 values corresponding to 3.44 and 6.84 mm, respectively.[132]
Berberine has gained considerable attention because of
its wide spectrum of biochemical and pharmacological
1713
O
O
O
N+
N+
O
O
OH
Coptisine
Pseudopalmatine
O
O
OH
O
H
Corydaline
Bulbocapnine
OH
O
HO
O
O
Corydine
2-Hydroxy-9-Methoxyaporphine
O
HN
H
OH
HO
O
O
(R,S)-2-N-Norberbamunine
O
O
N
H
O
O
O
HO
H
N
H
N
HO
(R,R)-Isochondodendrine
Figure 5
1714
(S-S)-O4-Methyl,O6-Demethyl(+)-Curine
Continued.
potentials. Its effects on AChE, BChE, MAO and other key

enzymes have been reported.[133,134] Additionally, berberine
can reduce Ab levels by altering APP processing in human
neuroglioma H4 cells, which stably express Swedish-type
APP in concentrations ranging from 0.1 to 100 mm.[135]
Molecular modelling studies have revealed that this molecule interacts with eight hydrophobic residues in AChE
and with six hydrophic residues in BChE. Also, there is the
possibility of a hydrogen bond between berberine and Tyr121 of AChE.[134]
The benzophenanthridine alkaloid chelerythrine has
been shown to inhibit AChE (from Electrophorus electricus
and humans) and BChE (from equine serum and humans)
in an equipotent way, with IC50 values ranging from 1.54
to 10.3 mm.[128] Kinetic studies indicated that it acts as a
mixed inhibitor of EeAChE and human AChE (hAChE)
with a competitive behaviour. Considering this mode of
inhibition, Brunhofer et al.[128] suggested that chelerythrine
could bind to the peripheral anionic site (PAS). Docking
simulations indicated that this molecule is able to bind in
the Torpedo californica AChE (TcAChE) (PDB ID 1FSS)
active site establishing a hydrogen bond with Tyr-130 and
p-stacking interactions with Tyr-121 and Tyr-334 PAS residues. This binding mode represents an interesting feature,
since it is possible to assume an additional capacity to
target the AChE-induced Ab aggregation process. Further
experiments demonstrated that chelerythrine at 5, 10 and
100 mm is able to inhibit the AChE-induced Ab fibrillogenesis in a concentration-dependent way reaching almost
90% of inhibition at the highest concentration.[128]
Beyond the previously cited berberine and palmatine,
another ten isoquinoline alkaloids from Corydalis saxicola
were tested on AChE.[127] The most active compounds, displaying IC50 values between 1.70 and 9.92 mm, were (-)-2,9dihydroxyl-3,11-dimethoxy-1,10-dinitrotetrahydroprotober
berine, (+)-nitroapocavidine, dehydrocavidine and sanguinarine. SAR studies indicated that all of the tested compounds were active at similar concentrations (IC50 values
close to 10 mm). AChE inhibition displaying similar IC50
values (110 mm) was also observed for other protoberberine alkaloids, such as jatrorrhizine, dehydrocorydaline,
pseudocolumbamine, pseudopalmatine
and
coptisine.[132,136,137] The activity-guided fractionation of Corydalis
cava led to the identification of three benzylisoquinoline
alkaloids inhibiting EeAChE and equine BChE (eqBChE):
bulbocapnine (EeAChE IC50 = 40 mm; eqBChE IC50 =
83 mm); corydaline (EeAChE IC50 = 40 mm; eqBChE
IC50 = 83 mm); corydine (EeAChE IC50 = 15 mm; eqBChE
IC50 > 100 mm).[138] These differences in potency and selectivity may be explained by the skeleton of the evaluated
alkaloids. Corydaline, which is most potent and selective for
EeAChE inhibition, is a protoberberine alkaloid, while the
other compounds possess the aporphine skeleton.[138]
Aporphine alkaloids from species belonging to Beilschmiedia displayed a narrow range of AChE inhibitory
activity with IC50 values in the range of 10-5 to 10-6 m,[139]
comparable with the reference compound huperzine A.
2-Hydroxy-9-methoxyaporphine, an alkaloid obtained
from B. alloiophylla, inhibited AChE activity with an IC50
in the region of 2.0 mm. On the other hand, compounds
possessing the aporphine skeleton isolated from Corydalis
turtschaninovii demonstrated lower potency, with
IC50 > 25 mm.[130]
The
bisbenzylisoquinoline
alkaloids
(R,S)-2-Nnorberbamunine, (R,R)-isochondodendrine and (S,S)-O4methyl,O6-demethyl-(+)-curine, isolated from Abuta
grandifolia (Menispermaceae), were able to inhibit AChE
from mouse brain and BChE from mouse serum, showing a
slight selectivity for the serum cholinesterase (about fivefold), with IC50 values ranging from 34.7 to 78.2 mm for
mouse AChE (mAChE) inhibition, and from 1.00 to
9.46 mm for mouse BChE (mBChE) inhibition.[140] This
inhibition profile is in agreement with that found for
other bisbenzylisoquinoline alkaloids from Cocculus
pendulus, another species belonging to the Menispermaceae
family.[141]
Indole alkaloids
Monoterpene indole alkaloids
The indole alkaloids nitrarine, hirsutine, rauwolscine and
catharanthine (Figure 6) display BChE selective inhibition,
with no significant difference between eqBChE and hBChE.
The IC50 values for eqBChE inhibition range from 4.97 to
10.6 mm, while the IC50 values for hBChE inhibition ranged
from 1.97 to 13.7 mm.[128] These results suggest that the
monoterpene side chain fused to the indole ring does not
critically influence the potential for BChE inhibition. In
agreement with these results, Passos et al.[142] verified that
the monoterpene indole alkaloids (MIAs) angustine, vallesiachotamine lactone and E/Z-vallesiachotamine also selectively inhibit eqBChE with IC50 values ranging from 3.47 to
14 mm (Table S3). Docking simulations indicated that vallesiachotamine lactone and E/Z-vallesiachotamine bind to the
BChE active site maintaining the same orientation of their
scaffold: the indole ring faces to Ser-198 and His-438 residues of the enzyme catalytic site, enabling the establishment
of hydrogen bonds. Moreover, the Trp-82, Trp-231, Leu286, Phe-329 and Ile-442 residues aid in stabilizing the
complexes through van der Waals contacts with the carbon
atoms characterizing the ring systems. Angustine, on the
other hand, binds to the BChE active site in a different orientation than vallesiachotamine structures. The complex
proteinligand seems to be stabilized mainly by hydrophobic interactions involving Trp-82, Trp-231, Leu-286 and
Phe-329 and the aromatic moieties of the molecule.[142]
1715
N
H
N
H
N
H
O
O
Nitrarine
Hirsutine
H
N
N
H
N
H
H
H
O
N
H
OH
O O
Catharanthine
Rauwolscine
O
N
N
H
N
H
H
O
Angustine
Vallesiachotamine lactone
N H
H
N
N
H
E-vallesiachotamine
Z-vallesiachotamine
N+
N+
N
H
N
O
Fascaplysin
Infractopicrin
N+
HO
H
N+
N
N
H
10-Hydroxy-infractopicrin
H
N+
N
H
1716
Prunifoleine
O
N+
Cl
N
H
14-Oxoprunifoleine
Figure 6
Nostocarboline
Indole alkaloids with anticholinesterase activity isolated from natural sources.

Quaternary b-carboline alkaloids

b-Carboline
alkaloids,
mainly
the
N3-alkylated
b-carbolinium salts, are well-known cholinesterase inhibitors. The marine compound fascaplysin was evaluated using
EeAChE and eqBChE and displayed IC50 values of 1.49 and
90.5 mm, respectively.[143] This inhibition profile suggests a
60-fold selectivity for AChE inhibition. In addition, kinetic
experiments demonstrated that fascaplysin acts as a
non-competitive EeAChE (Ki = 2.28 mm) and eqBChE
(Ki = 48.5 mm) inhibitor, indicating that this ligand binds
with high affinity to a site different from the catalytic
one.[143] Further docking studies revealed details regarding
the binding mode of this compound in AChE and BChE
active sites. Fascaplysin, which possesses a planar structure,
seems to occupy the AChE active site near to the gorge
entrance. The main interactions between the molecule and
AChE are pp interactions established between the terminal
aromatic ring of fascaplysin and the Trp-279 of the PAS.
Moreover, the carbonyl and NH groups may establish
hydrogen bonds with water molecules, which are conserved
in the enzyme active site. However, no direct hydrogen
bonds were observed to occur between ligand and amino
acids residues of the enzyme. Furthermore, hydrophobic
interactions can be observed between fascaplysin and the
residues Phe-330, Phe-331, Tyr-334 and Tyr-121.[143]
Two quaternary b-carboline alkaloids isolated from
the toadstool Cortinarius infractus, infractopicrin and
10-hydroxy-infractopicrin, were shown to selectively inhibit
bovine erythrocytes AChE (bAChE, IC50 of 9.72 and
12.7 mm, respectively). Neither compound showed significant inhibition on eqBChE when tested at 100 mm.[144] Based
on the chemical features of these two compounds, together
with findings of Schott et al.,[145] the authors assign the
AChE selectivity displayed by infractopicrin and 10hydroxy-infractopicrin to the indole N-alkylation (N1alkylation). Complementary docking studies were carried
out to investigate the selectivity for AChE inhibition. The
results revealed that both compounds may establish pp
interactions with the aromatic residues Trp-84 and Phe330. Furthermore, the carboxamide oxygen of the ligands is
connected with the hydroxyl oxigen of Tyr-121 through a
hydrogen bond, fixing their positions. On the contrary it
has been verified for AChE that the docking solution for
infractopicrin and 10-hydroxy-infractopicrin in BChE
shows no pp interactions between ligands and aromatic
residues and the lack of hydrogen bonds with Tyr-121.[144]
Prunifoleine and 14-oxoprunifoleine, two quaternary
b-carboline alkaloids found in Psychotria prunifolia, are able
to inhibit EeAChE with IC50 values of 10.0 and 3.39 mm,
respectively. Additionally, both alkaloids also inhibit
eqBChE with IC50 values of 100 and 11.0 mm, respectively.[142] As well as described for other quaternary
b-carbolines, they act as non-competitive inhibitors for

EeAChE (Ki of 15.8 and 8.93 mm, respectively). Further
docking experiments indicated that both molecules bind to
the TcAChE active site in the same orientation, insofar as
the endocyclic oxygen of both ligands is involved in a
hydrogen bond with the hydroxyl group of Tyr-121 from
the PAS.[142] Hydrophobic interactions occur between
ligands and Phe-290, Phe-330 and Phe-331 residues.
Moreover, stacking interactions between Trp-84 and the terminal aromatic ring of the two compounds are also established. These interactions are similar to those described in
earlier sections for Cortinarius infractus alkaloids[144] and for
some synthetic quaternary b-carbolines.[146] Docking simulations of prunifoleine and 14-oxoprunifoleine in hBChE
revealed that these compounds bind to the enzyme with different orientation, which can help to explain their different
potencies for eqBChE inhibition. In addition to hydrophobic interactions, prunifoleine, which is about 10-fold more
potent than 14-oxoprunifoleine, may establish polar contacts with Ser-198 and His-438 residues of the hBChE catalytic site.[142]
The cyanobacterial alkaloid nostocarboline is a nonselective AChE and BChE inhibitor displaying IC50 corresponding to 5.3 and 13.0 mm for EeAChE and eqBChE
inhibition, respectively.[147,148] The mode of inhibition, demonstrated by kinetic experiments, was non-competitive.[148]
Miscellaneous
Several other alkaloids have demonstrated anticholinesterase activity in addition to those mentioned above
(Figure 7). However, as they have a reduced number of representatives of their classes, these compounds have been
grouped together as miscellaneous alkaloids (Table S4).
Juliflorine is a piperidinium alkaloid isolated from the
leaves of Prosopis juliflora, and consists of two piperidines
connected with a dihydroindilizine moiety through two
aliphatic chains. This compound inhibited AChE with an
IC50 of 0.42 mm and BChE with an IC50 of 0.12 mm. Through
analysis by LineweaverBurk and Dixon plots, a noncompetitive type of inhibition was indicated. Docking
studies performed with TcAChE, suggested that C/D rings
enter deeper into the aromatic gorge, probably due to their
hydrophobicity, and that the rings A and B remain at the
top. The alkaloid is able to span the entire aromatic gorge,
with multiple binding sites and several intermolecular interactions identified, mainly hydrogen bonding, hydrophobic
contacts, pp interactions and hydrophilichydrophobic
interactions.[149] Some other piperidine alkaloids also inhibited AChE activity, although with lower potencies.[150]
From the Thai marine sponge Petrosia n. sp., petrosamine, a pyridoacridine alkaloid was isolated. This compound exists in both enolate and keto forms and showed a
1717
HO
B
N
H
HO
C
A
D
N
N
H
Juliflorine
Br
Br
2 3
4a
12b
11
N 9
8a
8
12c
4b
7a
OH
O
5
6
N+
14
13
12a
12
15
16
N+
O
Petrosamine
OH
N
N
N
N
Peganole
Figure 7
HO
Vasicine
Miscellaneous alkaloids with anticholinesterase activity isolated from natural sources.
potent inhibitory profile for AChE, with an IC50 of

0.091 mm. To understand the binding mode of petrosamine
to AChE, a docking study using TcAChE complexed with
galantamine and with donepezil was performed. It was
observed that the quaternary ammonium atom at ring D is
responsible for the major and strong interactions, mainly
pp interactions with Trp-84 and Phe-330 residues, and
by a salt bridge with Glu-199. Also, Ser-200 and His-440
residues from the catalytic site interact directly with
petrosamine.[151]
Three quinazoline alkaloids evaluated in a study by Brunhofer et al.[128] were shown to be selective BChE inhibitors.
1718
Desoxypeganine
Among them, peganole seems to be more selective for

eqBChE (IC50 = 11.4 mm) than for hBChE (IC50 > 20 mm).
On the other hand, vasicine showed similar potencies for
eqBChE (IC50 = 2.53 mm) and hBChE (IC50 = 3.13 mm) inhibition. Finally, unlike the other evaluated quinazoline alkaloids, desoxypeganine was a non-selective inhibitor, being
equipotent for eqBChE (IC50 = 11.8 mm) and eqAChE
(IC50 = 11.9 mm). The quinazoline scaffold has been used as
a model for the synthesis of hybrid compounds, which are
able to inhibit BChE in a range of nanomolar concentration, being about 80-fold more potent for serum cholinesterase inhibition than for AChE.[128]
Some other alkaloids with different skeletons, such as

leptomerine (IC50 = 2.5 mm)[152] and 8-demethoxy-10-Omethylhostasine (IC50 = 2.32 mm),[153] also may serve as good
scaffolds for AD treatment due to their high potencies.
Final remarks
Among the different alkaloid classes, most of the members
may be considered interesting anticholinesterase compounds. Steroidal and triterpenoidal alkaloids have, in
general, a slightly more selective potential as BChE inhibitors. Some of them exhibit an IC50 value lower than 1 mm
(e.g. buxakashmiramine, chonemorphine and chuanbeinone), all of them representing good scaffolds for the development of new BChE inhibitors. Some indole alkaloids,
such as angustine, vallesiachotamine and deoxyvobtusine,
are also more selective for BChE. However, several alkaloids
from other classes have only been evaluated for AChE activity, and when their inhibitory potential for BChE was
assayed, less selectivity was achieved. Although the role of
AChE in improving memory and cognition in patients with
AD is well established, the search for inhibitors of BChE,
acting in the CNS, should not be neglected, since this
enzyme is also responsible for inactivating acetylcholine in
the brain tissue, and its expression is increased in patients
suffering from AD.[154] Therefore, the evaluation of the
effects displayed by alkaloids on both cholinesterases could
constitute a valuable strategy for discovering new templates
for brain selective AChE/BChE inhibitors possessing less
undesired peripheral effects.
Considering the quinolizidine-type/Lycopodium alkaloids, except for huperzine A and B and sieboldine A, most
of the compounds from this class do not behave as potent
AChE inhibitors. Some of them, such as acrifoline, annotine
and annotinine, exhibit an IC50 value higher than 1000 mm,
which is a hindrance to their applicability as a source of
promising templates for cholinesterases inhibitors. On the
other hand, isoquinoline alkaloids remain good scaffolds
mainly as AChE inhibitors. The fact that the alkaloid galantamine, obtained from plants belonging to the Amaryllidaceae family, is one of the AChE inhibitors approved for
the treatment of AD has motivated the search of different
species within this family, aiming to identify other potent
cholinesterase inhibitors. Among these compounds, 1-Oacetyllycorine and sanguinine are potent AChE inhibitors
with IC50 values of 0.96 and 0.10 mm, respectively. Despite
the approximately 10-fold lower IC50 for AChE inhibition in
comparison with the value reported for galantamine, the
low natural abundance of sanguinine has precluded its
further development. Regarding the bioavailability, the
study of the interactions of Amaryllidaceae alkaloids with
the bloodbrain barrier efflux transporter P-glycoprotein
(P-gp)[155] indicated little or no interaction with P-gp for the
nine tested compounds, including galantamine. Moreover,

Amaryllidaceae alkaloids from the crinane and galantamine
groups did not show inhibitory activity against cytochrome
P450 3A4 (CYP3A4). The CYP3A subfamily comprises
about 30% of the total liver cytochrome P450 enzyme pool
in humans, and the isoenzyme CYP3A4 accounts for
approximately 60% of drugs metabolized. In the lycorane
series, only narciclasine inhibited CYP3A4 at low micromolar concentration.[156]
A number of the most active isoquinoline compounds
are quaternary alkaloids, such as berberine, palmatine, chelerythrine, coptsine and dehydrocorydaline. As such, these
compounds could have difficulty in crossing the blood
brain barrier under physiological conditions in a salt form.
Moreover, their oral bioavailability is usually poor, due to
their physicochemical properties.
It is worth noting that most of these studies only demonstrate the in-vitro activity of the isolated alkaloids, and it is
well known that other aspects, mainly pharmacokinetics
and toxicity, are related to the clinical potential of bioactive
molecules. Therefore, studies aiming to evaluate these
promising alkaloids in cell cultures and in in-vivo models
are indispensable. All these efforts should culminate in the
identification of good scaffolds and the subsequent development of more effective and safer anticholinesterase
drugs.
Conclusions and future perspectives

AD has a multifactorial pathology and although many
efforts are currently being devoted to achieving new
therapies aimed at reducing the amyloid and tauopathy
processes, the main therapeutic approach approved for
symptomatic treatment of AD is centred around the reduction of cholinergic deficit by the use of cholinesterase
inhibitors. Among these, the majority of studies have
focused on alkaloids, such as the prototype inhibitor of
AChE, physostigmine, obtained from Physostigma venenosum, which inspired the synthesis of the analogue rivastigmine. Another alkaloid currently marketed to treat AD
symptoms is galantamine, isolated from bulbs of several
Amaryllidaceae species. The major classes of molecules
reported to have significant anti-enzymatic activity are the
steroidal/triterpenoidal (most of them selectively inhibiting
BChE), quinolizidine (with focus on huperzine A, obtained
from the Chinese lycopod Huperzia serrata, currently
undergoing clinical trials), isoquinoline (especially galantamine type, protoberberine, aporphine and bisbenzylisoquinoline classes) and indole alkaloids, as summarized in
Table 1. Several active compounds have been discovered and
further investigated through molecular modelling studies,
to verify the sites of interaction between the chemical scaffold and the enzymatic amino acid residues, resulting in the
1719
Table 1 Main classes of alkaloids reported to have anticholinesterase activity, including their main enzymatic selectivity, mode of inhibition and the
IC50 range of concentrations for most of the representatives belonging to each class
Alkaloid class
AChE/BChE selectivity
Mode of inhibition
IC50
References
Steroids
BChE
Non-competitive[84]
[8597,107110]
Triterpenes
BChE
Non-competitive[105]
Quinolizidine
AChE IC50 ~10-710-3 M

BChE IC50 ~10-710-3 M
AChE IC50 ~10-610-3 M
BChE IC50 ~10-710-3 M
AChE IC50 ~10-610-3 M
BChE IC50 ~10-410-3 M
Isoquinoline
Amaryllidaceae
Benzylisoquinoline, Aporphine
AChE
AChE
AChE
Competitive[73]
[99104,106]
[73,81,111114,116,117]
Competitive[73]
Non-competitive/mixed
(chelerythrine)[128]
AChE IC50 ~10-710-3 M

AChE IC50 ~10-610-3 M
BChE IC50 ~10-710-3 M
[53,74,119125]
[126134,136141]
Indole
Physostigmine
AChE
Competitive[73]
[64,65]
MIAS
Quaternary b-carbolines
BChE
AChE
Non-competitive[143,146,148]
AChE IC50 ~10-7 M

BChE IC50 ~10-6 M
f
BChE IC50 ~10-610-4 M
AChE IC50 ~10-610-5 M
BChE IC50 ~10-510-4 M
[128,142]
[142148]
Despite some steroid and triterpene alkaloids displaying selectivity for AChE inhibition, most of compounds belonging to these classes present some
capacity for BChE inhibition. bResults refer to Huperzine A. cResults referd to galantamine. dThe Amaryllidaceae alkaloids possessing the galantamine
skeleton inhibit AChE with IC50 values close to 10-6 M. eIn general, the quaternary compounds are about five-fold more active for AChE inhibition
than the tertiary benzylisoquinoline alkaloids. fThe MIAs seem to inhibit both eqBChE and hBChE, displaying similar potencies (IC50 values ranging
from 1 to 15 mM).[128]
achievement of highly active and less toxic products. Currently, a great deal of work involving the development of
new anti-Alzheimer therapeutics is underway in order to
provide more efficient lead compounds for future use.
Funding
The review received no specific grant from any funding
agency in the public, commercial or not-for-profit sectors.
Declarations
Conflict of interest
The Author(s) declare(s) that they have no conflicts of
interest to disclose.
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Supporting Information
Additional
Supporting
Information
may be found in the online version of
this article at the publishers website:
Table S1 Structures of steroidal and triterpenoidal alkaloids with anticholinesterase activity

Table S2 Structures of quinolizidine
(Lycopodium) alkaloids with anticholinesterase activity
Table S3 Structures of isoquinoline and

indole alkaloids with anticholinesterase
activity
Table S4 Structures of miscellaneoustype alkaloids with anticholinesterase
activity
1725

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Alkaloids as a source of potential anticholinesterase

producing neuronal cell loss in the nucleus basalis of

Anticholinesterase alkaloids for AD

Eduardo Luis Konrath et al.

The second concerns neuroprotection, wherein oxidative

early in the course of AD, especially affecting the cholinergic

The amyloid hypothesis

Eduardo Luis Konrath et al.

of the neurotransmitter deficit observed in AD can be

t-Protein and neurofibrillary tangles

Anticholinesterase alkaloids for AD

with AChE,[36] but it does plays an important role in the

The use of cholinesterase inhibitors

Some cholinesterase inhibitors used to treat Alzheimers

Anticholinesterase alkaloids for AD

Eduardo Luis Konrath et al.

1993, providing cognitive improvement in 540% of

described.[50,51] Moreover, galantamine also improves

Eduardo Luis Konrath et al.

of its analogue rivastigmine, a synthetic compound with a

Anticholinesterase alkaloids for AD

aceae family, mainly in Buxus and Sarcococca genera, as

Anticholinesterase alkaloids for AD

Eduardo Luis Konrath et al.

Eduardo Luis Konrath et al.

Anticholinesterase alkaloids for AD

Anticholinesterase alkaloids for AD

Eduardo Luis Konrath et al.

into the aromatic gorge and is placed into the bottom,

rings.[107] As for the other steroidal alkaloids, the Fritillaria

Eduardo Luis Konrath et al.

Anticholinesterase alkaloids for AD

Quinolizidine/Lycopodium alkaloids with anticholinesterase activity isolated from natural sources.

Anticholinesterase alkaloids for AD

Eduardo Luis Konrath et al.

detected. Finally, the authors concluded that the lycopodane

Eduardo Luis Konrath et al.

Anticholinesterase alkaloids for AD

Isoquinoline-type alkaloids with anticholinesterase activity isolated from natural sources.

berberine alkaloids from Stephania venosa, verified that

five-fold more potent for EeAChE inhibition than the

Anticholinesterase alkaloids for AD

Eduardo Luis Konrath et al.

Eduardo Luis Konrath et al.

Anticholinesterase alkaloids for AD

displayed by the well-known quaternary alkaloids berberine

potency.[127132] Regarding BChE, both compounds inhibited

Anticholinesterase alkaloids for AD

Eduardo Luis Konrath et al.

Eduardo Luis Konrath et al.

potentials. Its effects on AChE, BChE, MAO and other key

Anticholinesterase alkaloids for AD

Anticholinesterase alkaloids for AD

Eduardo Luis Konrath et al.

Indole alkaloids with anticholinesterase activity isolated from natural sources.

Eduardo Luis Konrath et al.

Quaternary b-carboline alkaloids

Anticholinesterase alkaloids for AD

b-carbolines, they act as non-competitive inhibitors for

Anticholinesterase alkaloids for AD

Eduardo Luis Konrath et al.

Miscellaneous alkaloids with anticholinesterase activity isolated from natural sources.