European Journal of Medicinal Chemistry 70 (2013) 165e188

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European Journal of Medicinal Chemistry

journal homepage: http://www.elsevier.com/locate/ejmech

Invited review

Acetylcholinesterase inhibitors as Alzheimer therapy: From nerve

toxins to neuroprotection
Manjinder Singh, Maninder Kaur, Hitesh Kukreja, Rajan Chugh, Om Silakari,
Dhandeep Singh*
Pharmaceutical Chemistry Research Lab, Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala, Punjab 147002, India

a r t i c l e i n f o a b s t r a c t

Article history: Acetylcholinesterase is a member of the a/b hydrolase protein super family, with a significant role in
Received 14 May 2013 acetylcholine-mediated neurotransmission. Research in the modulators of AChEs has moved from a
Received in revised form potent poison (Sarin, Soman) in war times to the potent medicine (physostigmine) in peaceful times.
24 September 2013
Natural anti-AChE includes carbamates, glycoalkaloids, anatoxins derived from green algae; synthetic
Accepted 28 September 2013
anti-AChE includes highly poisonous organophosphates used as nerve gases and insecticides. Recently,
Available online 6 October 2013
the role of anti-AChE was reassessed from neurotoxins to neuron-protective in the diseases characterized
by impaired acetylcholine-mediated neurotransmission like Alzheimer’s disease (AD). So, the AChE has
Keywords:
Acetylcholine
been proven to be the most viable therapeutic target for the symptomatic treatment of AD. This review
Acetylcholinesterase article gives a spectrum of strategies to design AChE inhibitors used in the Alzheimer therapy.
Alzheimer’s disease Ó 2013 Elsevier Masson SAS. All rights reserved.
Nerve gases

1. Introduction
Research is motivated and funded mostly for the short term goals
which vary with time. Acetylcholinesterase (AChE) inhibitor physo-
stigmine was used in glaucoma in 1876 by physician Dr Ludwig
Laqueur, unknowingly about the mechanism of action of the drug [1].
Later on AChE inhibitors were utilized as pesticide and most of the
research was dedicated to its selective pesticidal action [2]. With the
eruption of war the selectivity of these agents was rather misused to
develop Sarin as the first nerve gas and later on during the war whole
research funding was concentrated on the nerve gases and finding
their antidotes. Hence, AChE inhibitors mainly phosphates as nerve
gas and oximes as antidotes came into the existence [3e5]. The post
war era saw the scarcity of food and hence the funding went into crop
growth and crop protection (pest management). The average popu-
lation age was less and growth was the need of the hour, hence this
era saw the development of pesticides for crops. Further, this era saw
the development of biological drugs (immunization) for longevity as
most of deaths accounted due to infections (bacterial, malaria etc.)
and many synthetic agents came into existence [6]. The major
breakthrough in the research came with the advent of recombinant
DNA technology and view of looking at a disease changed to a mo-
lecular level [7]. With an average population growing older the
prevalence of disorders of old age increased and hence in the present
era the major funding is targeted at older age disorders and hence
Alzheimer research came to front-stage. With concentration on Alz-
heimer, the history was revisited by Dr. Graeber in 1997 to study and
characterize the disease [8]. Along with AChE inhibitors, several other
therapies are also used for the management of Alzheimer’s disease
including Tau-based therapies, dealing with oxidative stress, target-
ing cellular Ca2þ handling, anti-inflammatory therapy, amyloid tar-
geted strategies (b-Secretase inhibitors and g-Secretase modulators).
Among these therapies, inhibition of b-Secretase causes the reduction
of Ab level along with blockage of all harmful downstream steps in the
pathogenesis of AD whereas g-Secretase modulators (GSMs) have
been shown to selectively lower Ab42 production without affecting
total Ab levels [9,10]. Although targeting amyloid seems to be favor-
able strategy but no BACE inhibitors or GSMs have reached market till
date. With the approval of an acetylcholinesterase inhibitor i.e.
Tacrine as an agent for Alzheimer by US-FDA, major funding and
research was concentrated in this thrust area [11]. This review high-
lights journey through time in the research of therapies targeted to-
wards Alzheimer with special emphasis on AChE inhibitors.
2. Alzheimer’s disease

Dementia is a loss of brain function that occurs with certain

* Corresponding author. Tel.: þ91 9814412412. diseases. Dementia usually first appears as forgetfulness. Dementia
E-mail address: maninder_688@yahoo.com (D. Singh). symptoms include difficulty with many areas of mental function

0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.09.050
166 M. Singh et al. / European Journal of Medicinal Chemistry 70 (2013) 165e188

Fig. 1. Schematic representation of journey of Alzheimer’s disease.

Fig. 2. Factors involved in Alzheimer’s disease progression.

including language, memory dysfunction, perception, emotional

behavior or personality and Cognitive skills (such as calculation,
was exclusively a rare pre-senile disorder [22]. In 1963, Terry
abstract thinking, or judgment). Most types of dementia are
studied histologic pattern of the tangles and plaque [23]. Plaques
nonreversible (degenerative). Nonreversible means the changes in
were interpreted to be: amyloid fibrillar core, surrounded by un-
the brain that are causing the dementia cannot be stopped or
myelinated dystrophic axons and dendrites containing filaments,
turned back [12,13].
dense bodies and paired helical filaments (PHF) [24]. In 1976, Dr
Alzheimer’s disease (AD) is the major cause of dementia, and is a
Robert Katzman reviewed the frequency and mortality of AD and
multifaceted neurodegenerative disorder characterized at a mo-
highlighted the need for focused research in this area [25]. As with
lecular level by protein misfolding and aggregation, oxidative
increased population age the prevalence of AD increased and was
stress, mitochondrial abnormalities, and neuroinflammatory pro-
bound to increase even further. Epidemiologists, clinical neurolo-
cesses [14,15]. It is characterized by a gradual onset and progression
gists, radiologists, psychiatrists, and psychologists quickly became
of deficits in more than one area of cognition, including episodic
active in developing better diagnostic methods for AD [26].
memory, mood and behavior changes, language, praxis and atten-
The prolonged history of scientific efforts to characterize better
tion, and the most common early symptom is difficulty in
the clinical features of dementia perhaps can be described in the
remembering newly learned information [16,17]. Brain area
context of six arbitrarily defined epochs (Fig. 1). Factors affecting
involved is the basal forebrain, cortex and amygdala, which are the
disease progression are summarized in Fig. 2 [27e29].
areas involved in learning, memory, attention and emotional
regulation. There are two forms of AD: Sporadic AD and Familial AD
4. Pathogenesis of AD
(FAD), Sporadic AD is characterized by a severe progressive decline
in cognition and increased neuronal cell death, and Familial AD
The exact cause of the Alzheimer’s disease is still uncertain, but
(FAD) develops much faster and is caused by mutations in com-
in general the following hypothesis has been put forward on the
ponents of the amyloid pathway such as Amyloid Precursor Protein
basis of the various causative factors (Fig. 3).
(APP), apolipoprotein E4 (ApoE4), presenilin-1 and presenilin-2
(PS1 and PS2) and sortilin-related receptor 1 (SORL1) [18,19].
4.1. Cholinergic hypothesis

3. History of AD Degeneration of neurons has been associated with loss of

AD is named after a German physician, Alois Alzheimer, who
first described it in the early 20th century on November 4, 1906 in
Tubingen (Wilkins, 1969). In 1901, Alois Alzheimer, a doctor at the
state asylum in Frankfurt, studied a patient Auguste D, 51-year-old
woman with symptoms of cognition and language deficits, auditory
hallucinations, delusions, paranoia and aggressive behavior. After
the death of the patient in 1906, Alois Alzheimer working with Emil
Kraepelin carried out the post-mortem of the brain and came to
know that her brain exhibited arteriosclerotic changes, senile pla-
ques, and neurofibrillary tangles and he subsequently published
the observations in 1907 [20]. In 1910, Kraepelin coined the term
‘Alzheimer’s disease’ e a term still used to refer to the most com-
mon cause of senile dementia.
In 1950s, increasing interest in theories and general ideas along
with the development of molecular biology lead to the formation of
genetic code concept [21]. In 1959, it was widely accepted that AD
memory function. The discovery of a link between the clinical
symptoms of the disease (memory loss) and specific cholinergic
deficits in the brains of people with AD, by Peter Davies in 1976, was
a landmark because it opened the door for modern neurochemistry
[30]. In this hypothesis deficiency of a critical neurotransmitter,
acetylcholine, in brain was observed either due to decreased pro-
duction of neurotransmitter or amplified acetylcholinesterase ac-
tivity [31]. This decreased level of the neurotransmitter causes
impairment of the cholinergic neurotransmission leading to the
loss of intellectual abilities. This hypothesis generally implies that
the cholinergic augmentation will improve the cognition in AD.
4.2. Amyloid hypothesis
Histological studies of the brain of the person with AD indicated
the presence of plaque, which lead to exclusive study of these ob-
jects. In 1984, building block of amyloidogenic peptide was found to
 M. Singh et al. / European Journal of Medicinal Chemistry 70 (2013) 165e188
Fig. 3. Schematic representation of AD pathogenesis in light of cholinergic, amyloid and tau hypotheses.
be amyloid beta protein that forms the amyloid fibrils in the
neuritic plaques. The amyloid cascade hypothesis considers that AD
is caused by the abnormal processing of b-amyloid [32,33].
In the amyloid hypothesis, a misfolded form of amyloid beta, an
oligomeric species, mainly toroidal or star-shaped deposited in the
brain may encourage apoptosis by physically piercing the cell
membrane. Plaque amyloid depositions or partially aggregated
soluble b-amyloid then start the neurotoxic cascade and causes
neurodegeneration that leads to AD [33,34]. Oxidative imbalance,
oxidative stress and functional changes in the production of b-
amyloid are the early steps of this disease [23]. The abnormal
metabolism of b-amyloids forms neurotoxic species due to mu-
tations in some of the components of the amyloid pathway, such
as APP, ApoE4, presenilin-1 and presenilin-2 (PS1 and PS2), and
SORL1 are responsible for autosomal-dominant early onset fa-
milial Alzheimer’s disease [35,36]. Therefore, inhibitors of b-am-
yloid aggregation appear as interesting candidates to treat AD in
its earlier phases [37,38]. In 1990s, the amyloid theory became a
powerful driving force that has dominated the direction of
research.
4.3. Tau hypothesis
In 1985, J.P. Brion and André Delacourte were the first to
suggest that tau might be the main component of neurofibrillary
tangles [39]. Soon after, in 1988, Michel Goedert and collabora-
tors cloned the cDNA of PHF-tau. Tau proteins, abundantly pre-
sent in neurons in the central nervous system, stabilize the
microtubules. In this process, hyperphosphorylated tau (the
altered protein) begins to couple with other threads of tau.
Eventually, they form neurofibrillary tangles inside nerve cell
bodies [40]. The formation of neurofibrillary tangles results in
disintegration of microtubules, collapsing the neuron’s transport
system [41]. This may lead to malfunctions in biochemical
communication between neurons and later results in the death
of the cells [42]. This is one of the expected reasons for the
deposition of the plaques in the brain.
167
4.4. Calcium hypothesis
The calcium hypothesis of brain aging and dementia began to
take shape in 1982. The original highly exploratory hypothesis
came into being in 1984 with little data or circumstantial evidence
to support. The role of activation of the amyloidogenic pathway in
remodeling the neuronal Ca2þ signaling pathways responsible for
cognition was explored by the calcium hypothesis of AD. Hydrolysis
of the APP yields two products that can influence Ca2þ signaling.
Firstly, the amyloids released to the outside form oligomers that
enhance the entry of Ca2þ that is pumped into the endoplasmic
reticulum (ER) further enhancing the sensitivity of the ryanodine
receptors (RYRs) to increase the amount of Ca2þ being released
from the internal stores. Secondly, the APP intracellular domain
may alter the expression of key signaling components such as the
RYR. This remodeling of Ca2þ signaling is proposed to result in the
learning and memory deficits that occur early during the onset of
AD. The fact that Ca2þ can either increase or decrease the strength
of central glutamatergic synapses complicates learning mecha-
nisms coordinated by neuronal calcium signaling systems. There is
a bidirectional relationship between Ca2þ signaling and the amy-
loidogenic pathway [43,44]. An increase in Ca2þ can stimulate the
metabolism of amyloid [45,46]. The amyloid metabolism results in
an upregulation of Ca2þ signaling by enhancing both the entry of
external Ca2þ and release of Ca2þ from the internal stores. This
upregulation of Ca2þ may account for both progressive decline in
memory and increase in neuronal cell apoptosis that occurs during
AD. As a result, the change in Ca2þ signaling in AD may switch the
brain from a system of memory storage to one of memory loss
(Fig. 4).
4.5. Isoprenoid change
The isoprenoid changes in Alzheimer’s disease differ from those
occurring during normal aging. During normal aging the human
brain shows a progressive increase in the level of dolichol and
reduction in level of ubiquinone but there is no change in
168 M. Singh et al. / European Journal of Medicinal Chemistry 70 (2013) 165e188
Fig. 4. Schematic representation of calcium hypothesis of AD.
concentration of cholesterol and dolichyl sulfate. In Alzheimer’s
disease, the situation is reversed with decreased levels of dolichol
and increased levels of ubiquinone. The concentrations of dolichyl
phosphate are also increased, while cholesterol remains un-
changed. The increase in the sugar carrier dolichyl phosphate may
reflect an increased rate of glycosylation in the diseased brain and
the increase in the endogenous antioxidant ubiquinone an attempt
to protect the brain from oxidative stress for instance induced by
lipid per oxidation [47].
5. Most promising target for the treatment of AD:
acetylcholinesterase
Acetylcholinesterase (EC 3.1.1.7; AChE) belongs to the a/b hy-
drolase fold protein super family; a group defined by common
structural homology and includes the cholinesterases, carbox-
ylesterases and lipases. Its principal physiological function is the
rapid hydrolysis of acetylcholine in the synapse and neuromuscular
junction, resulting in the termination of the nerve impulse [48]. The
three-dimensional structure of AChE has been first determined on
Torpedo californica (Tc) in 1991 via detailed analysis of the various
Fig. 5. Diagrammatic representation of active site of cholinesterase.
structural elements through a combination of structural studies
and site-directed mutagenesis [49,50]. Target enzyme AChE con-
sists of a narrow gorge with two separate ligand binding sites. In
medicine, AChE inhibitors are used mainly in the treatment of
Alzheimer’s disease (AD), glaucoma, neuromuscular blockade in
surgical anesthesia and myasthenia gravis [51]. In vertebrates, two
types of cholinesterase enzymes are present, Acetylcholinesterase
(AChE) and Butyrylcholinesterase (BuChE), both efficiently cata-
lyzes the acetylcholine hydrolysis. AChE is closely related to
butyrylcholinesterase (EC 3.1.1.8; BuChE). The two enzymes are
distinguished on the basis of substrate specificities, tissue distri-
bution and sensitivity to inhibitors [52,53].
5.1. Structure
AChE comprises mainly of two sites: catalytic triad and pe-
ripheral anionic site.
5.1.1. Catalytic triad
The AChE contains a catalytic triad (Ser200, His440 and Glu327)
located at the bottom of a deep and narrow gorge (about 20  A long
and as narrow as 4.5  A), lined with 14 aromatic residues (e.g.
decamethonium) [54]. The active site also contains a subside (the
“anionic subside”), including Trp84 as a key residue for the inter-
action with the quaternary ammonium group of the substrate
acetylcholine and other ligands via cation-p interaction, located
near the bottom of the cavity. Another conserved aromatic residue,
Phe330, is also involved in the interaction [55]. The cDNA sequence
indicates that there is a continuous stretch of 13 amino acids that
likely comprise a leader sequence that is missing in the native
enzyme. This peptide is rich in hydrophobic amino acids, which is
consistent with a putative membrane-spanning function. The
native enzyme contains a single polypeptide of 575 amino acids.
The AChE active site consists of three major domains: (1) an
esteratic locus (e.g. nerve agents), comprised of the active site
serine [56,57] (2) an anionic locus (e.g. tacrine) that is 14.7 
A from
the esteratic serine [58e60] and (3) a hydrophobic region that is
contiguous with or near the esteratic and anionic loci and that is
important in binding aryl substrates and active site ligands.
A fourth domain in the enzyme binds cationic ligands, such as
gallamine, D-tubocurarine, and decamethonium. With the help of
such developments in the field of structural determination of
acetylcholinesterase and the features of the catalytic triads leads to
design and development of novel AChE inhibitors as potential
therapeutic agents for the treatment of AD [61] (Fig. 5).
5.1.2. Peripheral anionic site (PAS)
The peripheral anionic site also known as b-anionic site (e.g.
aflatoxins, donepezil, huperzine) of AChE is not a well-defined area
that is located at the entrance of the catalytic gorge and is
approximately 14  A distant from the active centre [62e65]. Ligand
occupation of the peripheral anionic site frequently changes the
conformation of the active center [66,67] which contains Tyr 70,
Asp 72, Tyr 121, Trp 279 and Tyr 334. Among these amino acid
residues Trp 279 is a key residue, responsible for the adhesion
function of the enzyme AChE [48]. The aromatic site contains loops
and it has good conformational flexibility. The PAS binds to sub-
strate transiently as the first step in the catalytic pathway,
enhancing catalytic efficiency by trapping substrate on its way to
the active site. Amyloid b peptide interacts with the peripheral
anionic site resulting in the formation of amyloid plaques and
consequent damage to the cholinergic neurons [68]. The peripheral
anionic site is a target for a number of toxins and also promising
drugs [69,70]. The design of new AChEIs able to interact simulta-
neously with both the active and the peripheral site of the enzyme
 M. Singh et al. / European Journal of Medicinal Chemistry 70 (2013) 165e188
Fig. 6. Structures and names of G-type and V-type agents (in order of discovery).
AChE (dual binding site AChEIs) constitutes the main goal pursued
with the conjunctive approaches directed to the development of
new anticholinesterase agents with expanded pharmacological
profile, as a consequence of their higher affinity for the enzyme and
their interference in aggregation of b-amyloid through AChE pe-
ripheral site blockade [71]. AChE’s significance is in its being tar-
geted by a variety of anti-cholinesterases, ranging from snake
venoms to pesticides and the nerve gases [48].
5.2. Dual role of AChE in Alzheimer
The main stress of the cholinergic hypothesis is on the
enhanced activity of the enzyme acetylcholinesterase. The studies
have suggested that AChE is responsible for several non-catalytic
actions including the pro-aggregating activity of Ab. In individuals
having Alzheimer, the activity of the acetylcholinesterase in-
creases and leads to the augmented breakdown of the neuro-
transmitter acetylcholine and causes the decline in the
acetylcholine level in the brain. Another relation between the
enzyme and AD has been the partial involvement of the enzyme in
the formation of amyloid plaques and neurofibrillary tangles. It
has been shown that AChE promotes the aggregation of b-amyloid
peptide fragments by forming a complex with the growing fibrils.
These complexes have been shown to be more cytotoxic than b-
amyloid fibrils alone [72,73]. A structural domain of AChE that
promotes b-amyloid peptide fibril formation has been identified
to be the peripheral anionic site of the enzyme [74,75]. The mol-
ecules that interact either exclusively with PAS or with both cat-
alytic and peripheral binding sites of AChE prevent the pro-
aggregating activity of AChE toward Ab. Furthermore, studies
have also revealed that several AChE inhibitors not only facilitate
cholinergic transmission, but also interfere with the synthesis,
deposition and aggregation of toxic Ab. Thus, AChE inhibition has
been documented as a critical strategy for the effective manage-
ment of AD. Accordingly, compounds showing dual binding with
the AChE, that is, with catalytic and peripheral sites represent new
therapeutic agents for treatment of AD.
AChE is a sensitive target for both natural and synthetic
cholinergic toxins. Among the natural anti-AChEs are plant-derived
carbamates and glycoalkaloid inhibitors [76]. A natural inhibitor of
AChE was also found in a mollusk [77]. Blue-green algae is
169
equipped with anatoxins, highly effective toxins that block the
active site [78,79]. Green mamba venom includes the neurotoxic
peptide fasciculin, which blocks the entrance to the active and the
peripheral sites of AChE [80]. Use of anti-AChEs as defense and
attack weapons in nature, therefore, preceded their use by humans.
Synthetic anti-AChEs were first studied and manufactured as highly
poisonous organophosphate and carbamate nerve gases and in-
secticides. In the clinic, controlled use of AChE inhibitors has proved
valuable for the treatment of diseases that involve compromised
acetylcholine-mediated neurotransmission.
5.3. Journey of AChE inhibitors
5.3.1. Pre-war era
Nerve agents (NAs) act by interacting with the enzyme acetyl-
cholinesterase via phosphorylation, leading to CVS, CNS, respira-
tory failure and seizures [81]. Examples of NAs include tabun, Sarin,
Soman, cyclosarin, and VX [82e84]. Peripheral manifestations of
nerve gas includes wheezing, cough, dyspnea, sweating, salivation,
nausea, vomiting, diarrhea, hyperglycemia, metabolic acidosis,
ketosis, atrioventricular blocks, hypotension and Central Nervous
System Manifestations includes headache, dizziness, impaired
memory, anxiety, tension, emotional instability, lethargy, ataxia,
seizures, respiratory depression, severe muscle contractions fol-
lowed by paralysis [85]. The mechanism of action, i.e. cholines-
terase inhibition, was discovered during World War-II by German,
English and US scientists, the data was published only after the War
[86]. NAs inhibit AChE by making reversible complex which is
reactivated by recommended antidotes including atropine,
scopolamine, pralidoxime chloride, and anticonvulsant medica-
tions [87]. Oximes, such as pralidoxime chloride, act as chemical
reactivators of inhibited AChE and must be administered to all
patients after NA exposure to overcome their effects. Oximes can
reactivate bound AChE by removing the OPC from the OPCeAChE
complex [88].
Historically, the synthesis of the first potentially lethal OPC,
tetraethyl pyrophosphate, occurred in 1854 in the laboratory of De
Clermont in France. In the 1930s, major research funding was
concentrated on production and scaling of the nerve gases. The
highly toxic OPCs Tabun, Sarin, and Soman were developed during
pesticide research by the Germans [89]. In 1934, a project on
170 M. Singh et al. / European Journal of Medicinal Chemistry 70 (2013) 165e188
Fig. 7. Structures and names of organophosphate-based pesticides.
synthetic insecticides was started at I.G. Farben Industrie (Ger-
many) by Otto Bayer who assigned all further research to the
chemist Gerhard Schrader. In 1936, the German chemist Gerhard
Schrader, reasonably working on insecticides, developed an
extremely effective organophosphate insecticide called tabun [90].
In 1937, a sample of tabun was sent to the Ministry of War and
further research determined that tabun exerted its effect by inter-
fering with nerve transmission. Thus, the G-series NAs were born.
Schrader was given a secret lab to continue research and devel-
opment. In 1938, Schrader synthesized Sarin. In 1940, secret con-
struction began on multiple pilot plants for small-scale tabun
production. In 1944, German scientist and Nobel Laureate (Chem-
istry, 1938) Richard Kuhn discovered the nerve agent Soman
[91,92]. NAs were first produced by German scientists during WWII
and were known by German code names: GA (tabun), GB (Sarin),
GD (Soman), GF (cyclosarin), and VX. In 1952, the compound VX
(venom compound X) was formulated. VX (V for venomous) was
discovered by the British and produced by the Americans after
World War II (WWII) [93]. It is the most lethal of all the NAs.
Structures and names of G-type and V-type agents has been shown
in Fig. 6.
5.3.2. Post war era
After the war the twentieth century population saw the scarcity
of food and the funding went into crop growth and crop protection
(pest management). The average population age was less and
growth was the need of the hour, hence this era (the 1940s and the
1950s) saw the development of pesticides for crops and was named
as the pesticide era [94].
To dispose of pests (i.e. mainly insects) by chemical means the
preparation of pesticides is entangled with that of the nerve agents
since they possess similar structural features and formulas. The
mode of action is same as nerve agents, by inhibition of AChE, the
enzyme responsible for breaking down acetylcholine (Ach) which
is a neurotransmitter found at neuromuscular junctions but are less
hazardous [95]. Organophosphates cause a delayed neuropathy
that has been termed organophosphate-induced delayed neuro-
toxicity (OPIDN). OPIDN is a progressive neurological condition
characterized by weakness, ataxia and subsequent paralysis of the
limbs [96,97]. Most AChE-inhibiting pesticides are divided into two
different categories, organophosphates and carbamates; they differ
in the reversibility of their effects. Carbamates like carbofuran
temporarily inhibit AChE [98].
 M. Singh et al. / European Journal of Medicinal Chemistry 70 (2013) 165e188
Fig. 8. Timeline history of acetylcholinesterase inhibitors.
Before the 1930s, pesticides took the form of chlorinated organic
compounds. As with related chemical warfare developments,
chlorine-containing pesticide compounds eventually gave way to
phosphorus compounds. The development of organophosphate-
based pesticides (insecticides) after World War II (1940se1950s)
synthesized second generation of compounds including chlorpyr-
ifos, coumaphos, cyanophos, demeton, demeton-S-methyl, diaz-
inon, dichlorvos, dioxathion, glyphosate, fonofos, malaoxon,
malathion, methamidaphos, mevinphos, oxydemeton-methyl,
paraoxon, and parathion. Nerve agents differ from pesticides in
terms of much greater potency of the nerve agents whereas pes-
ticides possess longer duration of the biological effects [99].
Structures and names of selected relevant organophosphate-based
pesticides are shown in Fig. 7 [100].
The growth in synthetic pesticides accelerated in the 1940s with
the discovery of the effects of DDT, BHC, aldrin, dieldrin, endrin,
chlordane, parathion, captan. In 1946 resistance to DDT by house
flies was reported [101]. Organophosphorous compounds have
importance to those concerned with military as well as with agri-
cultural matters. These are too toxic that they kill the mammals
much more readily than they would kill insects. Common symp-
toms associated with Carbamate and Organophosphate Pesticide
are poisoning fatigue, headache, dizziness, blurred vision, excessive
sweating/salivation, nausea/vomiting, stomach, cramps, and diar-
rhea, inability to walk, weakness, chest discomfort, constriction of
pupils, unconsciousness, muscle twitching, running nose, drooling,
breathing difficulty, coma and death [99].
Under pressure of World War II, the pharmaceutical manufac-
turers rapidly adapted mass production methods for the antibiotics.
The second quarter of the 20th century marked the flowering of the
antibiotic era: a new and dramatic departure in the production of
disease-fighting drugs [102]. Fleming’s discovery of penicillin in
1929 went undeveloped and Florey and Chain studied it in 1940.
Antibiotic discoveries came rapidly in the 40’s. In the Pre-Antibiotic
Era prior to the 1940’s millions of people died from common
171
bacterial infections. In the last 50 years all the bacterial infections
can almost always be cured by using antibiotics [7].
When, in 1894, Behring and Roux announced the effectiveness
of diphtheria antitoxin, pharmaceutical scientists both in Europe
and in the United States rushed to put the new discovery into
production [103]. Parke, Davis & Company was among the pioneers.
The serum became available in 1895, and lives of thousands of
children were saved. Inoculation of horses with diphtheria toxin
was the first step of many in producing antitoxin. In 1903, Parke-
Davis received U.S. Biological License No. 1. New improved biolog-
ical products have continued to become available, climaxed in 1955
by poliomyelitis vaccine [104].
Pharmacological inhibitors of AChE are important in controlling
diseases that involve impaired acetylcholine-mediated neuro-
transmission. Use of anti-AChEs as defense and attack weapons in
nature, therefore, preceded their use by humans. Synthetic anti-
AChEs were first studied and manufactured as highly poisonous
organophosphate and carbamate nerve gases and insecticides. In
the clinic, controlled use of AChE inhibitors has proved valuable for
the treatment of diseases that involve compromised acetylcholine-
mediated neurotransmission. For example, Alzheimer’s disease
involves selective loss of cholinergic neurons in the brain [105]. In
myasthenia gravis, auto-antibodies reduce the number of nicotinic
acetylcholine receptors at the neuromuscular junction [106]. AChE
inhibition increases the synaptic concentration of acetylcholine and
allows a higher occupancy rate and longer duration at its receptor
[107]. Nevertheless, anti-AChE therapeutics do not address the
etiology of the diseases for which they are used.
The twentieth century was marked by an incredible rise in life
expectancy and an equally impressive decline in infant mortality in
the developed world. Prosperous people live longer and old age
carries a high risk of dementia, a condition that is so far neither
preventable nor curable. Alois Alzheimer described the Alzheimer
disease in 1907, but it was not until 60e70 years later that any new
significant developments were reported on the pathology of this
172 M. Singh et al. / European Journal of Medicinal Chemistry 70 (2013) 165e188

Table 1
Structures of acetylcholinesterase inhibitors.

tbl1
Compound no. Structure

Fig. 9. Natural flavones used in AD.

disease. The number of laboratories involved and the pace of

research on AD remained quite slow till the 1980s. Drug discovery
is a time process involving target site identification, followed by
4
validation of target, drug’s ability, designing a molecule, testing in-
vitro, in-vivo, toxicity and safety analysis. All this process is a
laborious process. In the year 1976, Peter Davies proved that the
major cause of Alzheimer was the loss of Acetylcholine neuron
causing deficiency of ACH in the brain. The link between decreasing
levels of acetylcholine and Alzheimer’s disease has been estab-
lished due to the purpose of acetylcholine with regards to memory 5
and Alzheimer’s being a disease related to loss of memory. Centrally
acting agents were sought after for the treatment to increase the
level of Ach in the brain. Hence, AChE became a target for AD.
Pioneering research in the field of Nerve Agents and Pesticides had
already been carried out at mass level with maximum funding
concentrated on research in inhibition of AChE. This background
research work made it possible to get the first drug for AD
belonging to this class and four approved drugs belong to this class 6
as well (Fig. 8).
In the mid-1980s, office of Alzheimer’s disease, established by,
the Director of NIA, T. Franklin Williams, with the support of NIH,
Director James Wyngaarden joined NIA in AD research. In 1984,
development of national, interdisciplinary research program spe-
cifically focused on the causes and the leaders at the NIA decide the
course of AD and the differences between AD and normal aging. In
7
1986, Summers published the first results obtained with tacrine (1)
in the New England Journal of Medicine [108]. It was the first-
approved drug among the first generation cholinesterase in-
hibitors. New, well-tolerated and more efficient second-generation
anticholinesterase inhibitors, such as donepezil (1996), and riva-
stigmine (1998), subsequently made their appearance; later in
2000, galantamine was brought out. The use of memantine, an
NMDA receptor antagonist, to treat patients in moderate to severe 8
stages of the disease was approved in 2002 [109]. Various natural
derivatives such as flavones including Apigenin, Oroxylin A,
Luteolin, have marked effect on central nervous system to improve
memory and cognition, hence beneficial in the treatment of AD
[110] (Fig. 9).

5.4. Classification of acetylcholinesterase inhibitors 9

Various natural, semisynthetic, and synthetic derivatives for AD

treatment have been synthesized and can be broadly classified
based on chemical structures. The majority of drugs for AD
 M. Singh et al. / European Journal of Medicinal Chemistry 70 (2013) 165e188 173

Table 1 (continued ) Table 1 (continued )

Compound no. Structure Compound no. Structure

10 17

18

11

19

12

20
13

14 21

22

23
15

24

16 25

26

(continued on next page)

174 M. Singh et al. / European Journal of Medicinal Chemistry 70 (2013) 165e188

Table 1 (continued ) Table 1 (continued )

Compound no. Structure Compound no. Structure

37
27

38

28

39

40
29

41a

30

41b

31

42

32

33

43

34

44
35

HO
N
O CH3
H3C
45 N
36

MeOOC
 M. Singh et al. / European Journal of Medicinal Chemistry 70 (2013) 165e188 175

Table 1 (continued ) Table 1 (continued )

Compound no. Structure Compound no. Structure

46

53c

47

48 54

49
55

50

56

51
57

52
58

59

53a

60

53b

61

(continued on next page)

176 M. Singh et al. / European Journal of Medicinal Chemistry 70 (2013) 165e188

Table 1 (continued ) Table 1 (continued )

Compound no. Structure Compound no. Structure

62

69

63

70

64

71

65

72

73
66

74
67

75

68

76
 M. Singh et al. / European Journal of Medicinal Chemistry 70 (2013) 165e188 177

Table 1 (continued ) Table 1 (continued )

Compound no. Structure Compound no. Structure

77

88

78

89

79

90

80
91

92

81

93

82

94
83

84

95

85

96
86

87
97

(continued on next page)

178 M. Singh et al. / European Journal of Medicinal Chemistry 70 (2013) 165e188

Table 1 (continued ) Table 1 (continued )

Compound no. Structure Compound no. Structure

109
98

treatment focuses on the cholinergic hypothesis. Various studies

99
have been conducted on the designing of new highly efficient and
active derivatives of already existing FDA approved drugs.

5.4.1. Tetrahydroacridine derivatives (tacrine (1,2,3,4-

tetrahydroacridine)) analogs
100 Tacrine (1, Table 1) was the first drug approved for treatment of
AD in 1993 [111]. It is a potent inhibitor of both AChE and BuChE. Its
structure has been modified on the basis of importance of 4-
aminopyidine motif in the tetrahydroacridine system.
4-Aminopyridyl moiety in the tetrahydroacridine group was
found to be important for the potent activity. The modifications,
categorized into heterocyclic ring modification and 4-amino sub-
101
stitution, can be targeted keeping 4-aminopyridyl moiety
conserved.

5.4.1.1. Five membered heterocyclic ring derivatives. It includes the

modification of either ring A or ring C, whereas ring B remains
intact.
102
5.4.1.1.1. Substituted pyrazole derivatives. These derivatives in-
cludes the replacement of the ring A of tacrine nucleus with the
substituted pyrazole ring, structurally planned on basis of the
bioisosteric relationship between the quinoline ring, included in
the 1,2,3,4 tetrahydroacridine system of THA and the azaheter-
ocyclic pyrazolo [3,4-b]pyridine system [112].Compound 2, bearing
a phenyl group in position 3, showed the best activity (Table 1).
103 5.4.1.1.2. Furan derivatives. These derivatives include the
replacement of the ring A of doctrine by substituted furan ring
systems. Newly derived molecules showed the acetylcholinesterase
inhibitory potential with the high degree of selectivity toward the
acetylcholinesterase (Compounds 3 and 4, Table 1) [113].
5.4.1.1.3. Thieno derivative. These derivatives involve the
104
repalcement of ring A with the substituted thiophene ring, but
these compounds were found to possess little inhibitory activity
against the acetylcholinesterase [114].

5.4.1.2. Six membered substituted heterocyclic ring derivatives.

105 The ring A can also be replaced with the substituted six membered
heterocyclic rings.
5.4.1.2.1. Pyridine substituted derivatives. These series of com-
pound comprises of modification in both A and C ring, involves the
replacement of the ring A of tacrine with the substituted six
106 membered heterocyclic ring i.e. substituted pyridine, along with a
change in the size of the C ring of tacrine. Out of the number of
derivatives synthesized, the compound 5 and 6 (n ¼ 1, 2) (Table 1)
possess highest acetylcholinesterase inhibitory potential [115,116].
5.4.1.2.2. Substituted pyran derivatives. These derivatives result
107 from replacement of the ring A of tacrine with the substituted
pyran, a heterocyclic aromatic ring system [116].Out of the various
synthesized substituted pyran derivatives, derivatives 7 and 8
(Table 1) have shown the highest acetylcholinesterase inhibitory
potential with IC50 values of 8.68  107 M and 1.82  107 M
respectively.
108
5.4.1.3. Fused heterocyclic rings analogs. These derivatives include
the replacement of ring A of tacrine with the fused heterocyclic
 M. Singh et al. / European Journal of Medicinal Chemistry 70 (2013) 165e188
rings, one of the ring system used is pyrazolo [3,4-b]pyridine [113].
The compound 9 have shown good inhibitory potential against
acetylcholinesterase with IC50 value of 6.4 mM (Table 1).
5.4.1.4. Amino substituted derivative
5.4.1.4.1. Imidazole substituted derivatives. These derivatives
include substitution of substituted imidazole ring at the amino
group of the 4-aminopyridine ring of tacrine. This kind of modifi-
cation possess additional abilities such as inhibition of histamine
metabolism, N-methyltransferase (NMT) and histamine H3 recep-
tor antagonism which can improve reduced cognitive functions
along with acetylcholinesterase inhibition [117,118].One of the
compound (10) among these designed molecules possesses good
AChE inhibitory potential (Table 1).
5.4.1.4.2. Piperidine substituted derivatives. The amino group of
tacrine has also been substituted with the piperidine, both the ar-
omatic nucleus are separated by the alkyl or an oxyphenyl group in
the tether and led to the design of the most effective compound 11
(Table 1) possessing good acetylcholinesterase inhibition [117,118].
5.4.1.4.3. 1,2-Dithiolane derivative. In these derivatives, amino
group has been substituted with 1,2-dithiolane, five membered
heterocyclic ring. The most common derivative is the tacrine-lipoic
acid dimer. Lipoic acid (universal antioxidant) has been combined
with a tacrine moiety by a carbon chain of varied length (2e7
methylene groups). The most potent derivative with three methy-
lene group linker, compound 12, showed IC50 of 6.96 nM (Table 1).
Along with acetylcholinesterase inhibition, it also shows an addi-
tional antioxidant property [119].
5.4.1.4.4. Heteroarylpiperazine substitution. Besides acetylcho-
linesterase inhibition, these moieties also exhibit antiemetic ac-
tivity via producing 5-HT-3 agonism. The 5HT-3 organism can be
possibly due to the 2-piperazinylquinoline-4-carboxamide
attached to the tacrine ring. Compound 13 (Table 1) has been
found to possess the optimum activity with IC50 ¼ 4.1 nM against
human AChE [119].
5.4.1.4.5. Tacrine dimers. Homodimers of two tacrine moieties
connected by oligomethylene chains of different lengths have also
been designed [120]. These compounds are more hydrophobic than
THA because of the introduced alkylene chain that interacts
simultaneously with the catalytic site and the peripheral anionic
site. Among them, a compound containing two tacrine subunits
whose amino groups are connected by an heptamethylene chain,
14, which was designed taking into account the existence of two
binding sites for tacrine in AChE, is about 1000 times more potent
than tacrine, although its toxicity is not known yet (Table 1).
5.4.1.4.6. Melatoninetacrine hybrids. Amino group has also been
substituted with the melatonin and the hybrid so formed have been
reported to possess AChE inhibitory potential, strong antioxidant
actions and is able to directly scavenge a variety of reactive oxygen
species [120].Compound 15 possess the optimum activity with IC50
value of 2 nM (Table 1).
5.4.1.4.7. Tacrine-drugs dimers. The substitution at amino group
of tacrine with other drugs of AD like Donepezil (18) produces
Tacrine and Donepezil related Hybrid (Table 1). Out of the various
synthesized hybrid compounds two compounds 16, 17 possess the
optimum AChE inhibitory potential [121]. All of the new compounds
demonstrated significant inhibition of beta-amyloid aggregation
and were shown to be more potent than parent compounds.
5.4.2. N-Benzylpiperidine derivatives
Donepezil (18) was approved in 1996 for the treatment of mild-
to-moderate AD. It has been designed as AChE bivalent inhibitor.
The basic structure of donepezil has been modified and the effect
on the inhibitory potential of the compounds was observed. Ring of
donepezil has been kept intact as such while indanone ring of the
179
donepezil has been substituted with the other heterocyclic ring
systems [122].
5.4.2.1. Benzisoxazole substituted derivatives. These derivatives
worked on the replacement of indanone ring of Donepezil with
benzisoxazole ring system. Among the synthesized N-benzylpi-
peridine-benzisoxazole derivatives, compound 19 (Table 1)
exhibited the potent AChE inhibitory activity with IC50 value of
0.8e14 nM [123,124].
5.4.2.2. 1,2,4-Thiadiazolidinone substituted derivatives. These de-
rivatives involves replacement of indanone system of donepezil
with 1,2,4-thiadiazolidinone ring [124].The compound 20 pos-
sesses activity profile comparable with tacrine instead of donepezil
(Table 1). It also displayed more selectivity toward the AChE
(IC50 ¼ 0.14 mM).
5.4.2.3. Benzylpiperidinone substituted derivatives. These de-
rivatives involve the replacement of indanone system of donepezil
with substituted indole or pyrrole ring [125]. The compound 21
(Table 1) possess the AChE inhibitory potential (IC50 ¼ 8 mM).
5.4.2.4. Aroyl(thio)urea substitution. These compounds involve the
replacement of indanone ring with aroyl (Thio) urea system while
the N-benzylpiperidine ring system was kept intact. The compound
22 (Table 1) exhibited an in vitro nanomolar AChE inhibitory activity
and in vivo it reversed scopolamine-induced amnesia in the passive
avoidance paradigm in rats with dose 1000-fold lower than its LD50.
5.4.2.5. Other N-benzylpiperidine derivatives. TAK-147 (23) and
TAK-802 (24) have been developed by the Takeda Chemical In-
dustries (Table 1). Preliminary SAR studies concluded that the
introduction of an additional ring in the structure of TAK-147 (23)
caused an increase in the biological activity whereas further
introduction of the hydroxy and fluoro groups results in decreased
activity [126].
5.4.3. Benzophenone derivatives
These compounds encompass an aromatic function and a ter-
tiary amino moiety connected by a suitable spacer. In particular, the
benzophenone nucleus as aromatic function and the N,N-benzyl
methylamine as tertiary amino function has been selected. Com-
pound 25 and 26 possess optimum AChE inhibitory potential
(Table 1) [127].
5.4.4. Di-benzofuran analogs
Rivastigmine is a small molecule that easily crosses the blood
brain barrier and possesses BuChE and AChE inhibitory properties.
It (27) was approved in 2000 for the treatment of mild-to-moderate
AD (Table 1). In 2007, it was reformulated for delivery through a
transdermal patch. This has resulted in significantly lower GI side
effects compared to the oral capsule.
Various derivatives of rivastigmine have been synthesized but
the compound 28, i.e. the 5-thia-1-azacyclopenta[a]naphthalene
derivative, exhibited the highest activity (Table 1). Replacement of
the methyl chain with an ethyl chain results in 14e23 fold
decreased AChE inhibitory activity. The alkyl substituent on the
carbamoyl nitrogen determined the profile of activity. Replacement
of the methyl group in the carbamoyl moiety by a 1-phenylethyl
substituent caused significant decrease in inhibitory activity to-
wards both AChE and BuChE [128].
5.4.5. Galantamine analogs
Galantamine was approved for the treatment of mild-to-
moderate AD in February 2001. Galantamine (29), an alkaloid
180 M. Singh et al. / European Journal of Medicinal Chemistry 70 (2013) 165e188
isolated from the Caucasian snow-drop (Galanthus woronowii) and
from the bulbs of different species of the Amaryllidaceae family, is a
centrally acting, selective, reversible, and competitive acetylcho-
linesterase (AChE) inhibitor, as well as an allosteric modulator of
the neuronal nicotinic receptor for acetylcholine [129e131]. Gal-
antamine has also been shown to enhance g-aminobutyric acid
(GABA) and glutamate release in hippocampus slices (Table 1)
[132].
5.4.5.1. Open D-ring galantamine analogs. New optically pure open
D-ring galantamine analogs have been synthesized [133] and the
most active inhibitor was found to be compound 30 (Table 1).
5.4.5.2. N-Hexyl-benzyl piperidine substituted derivatives.
Structure activity relationship (SAR) studies reveal that substitution
on the nitrogen atom of galantamine has been favorable for AChE
inhibitory activity, may be the substituent display interaction with
the peripheral anionic site (PAS). N-substituted galantamine de-
rivatives designed by selecting benzyl amino groups and modified
benzyl amino moieties (such as amide group) as pharmacophoric
units and incorporating them into the galantamine molecule for
better AChE inhibitory activity. Besides, different lengths of the
alkyl chain between galantamine and benzyl amino moieties has
been explored, the compound 31 (Table 1) was observed to be the
most active [134].
5.5. Natural compounds exhibiting acetylcholinesterase inhibitory
potential
5.5.1. Alkaloids and related compounds
5.5.1.1. Physostigmine and its analogs. Physostigmine, an alkaloid
isolated from Physostigma venenosum, possesses AChE inhibitory
activity. Physostigmine may penetrate the CNS, so the development
of physostigmine derivatives has been undertaken. This resulted in
obtaining additional lipophilic analogs of physostigmine such as
phenserine and geneserine [135e137]. The first medical treatment
of glaucoma was introduced by Ludwig Laqueur, Professor of
Ophthalmology at Strassburg, who found that physostigmine
(eserine) would lower tension in glaucomatous eyes. He began his
studies on the drug in 1875. In 1876, he personally learned about
the hypotensive effect of physostigmine [138]. It was the first AChE
inhibitor investigated for the treatment of AD. It is isolated from the
seeds of P. venenosum.
Some geneserine derivatives with a substituted phenyl ring
attached to the carbamoyl nitrogen atom have been synthesized
[139]. The most active compounds of the series were 32 and 33
(Table 1).
Several potent cholinesterase inhibitors in the series of phen-
serine and geneserine analogs have been identified. Phenserine
derivatives containing one, two or three methyl groups at different
positions of the phenyl ring have been synthesized. In the group of
mono-substituted derivatives, the compound with a 20 -methyl
substituent (34) has been the most active and selective inhibitor of
AChE (Table 1). Compounds with a disubstituted phenyl ring
exhibited similar activity with selectivity for AChE but tri-
substituted derivatives were found to be inactive [140]. Phenser-
ine has a dual effect: decreasing beta-amyloid precursor protein
and has a reversible AChE inhibition and has reached Phase III
clinical trials (2003e2004).
Tolserine another analog of physostigmine differs from phen-
serine at the 2-methyl substitution on its phenylcarbamoyl moiety.
Preclinical studies were initiated in 2000, and it was shown to be
200-fold more selective against hAChE versus BuChE [141]. Tol-
serine proved to be a highly potent inhibitor of human AChE
compared to its structural analogs physostigmine and phenserine.
In the structure of physostimine, replacement of the nitrogens in
the pyrrolodine ring with oxygen led to new derivatives [142]. The
AChE inhibitory activity of enantiomers of tetrahydrofur-
obenzofuran and methanobenzodioxepines has also been reviewed
[143]. It has been revealed that (3aS) tetrahydrofurobenzofurans
(35) and (5S)-methanodioxepines (36) analogs were potent AChE
inhibitors (Table 1).
5.5.1.2. Piperidine alkaloids. New piperidine alkaloids: ()-3-O-
acetyl-spectaline and ()-spectaline have been obtained from the
flowers of Senna spectabilis sin. Cassia spectabilis, Leguminoseae
[144e147]. An acetylcholine fragment, which could be recognized
in the structure of these alkaloids, prompted to design and syn-
thesize new semi-synthetic piperidine alkaloids with expected
AChE inhibitory activity. Compounds 37 and 38 have found to be
effective inhibitors of rat brain tissue AChE and weak inhibitors of
BuChE (Table 1). These compounds displayed selectivity towards
brain AChE [144] and showed mean IC50 value of 7.32 and 15.1 mM,
respectively.
5.5.1.2.1. Huperzine derivatives. Huperzine A and B are alkaloids
isolated from the Chinese herb Huperzia serrata, which is a member
of the Lycopodium species, used in traditional herbal medicine. It
induces significant improvement of memory in aged subjects and
patients with Alzheimer’s disease. Both alkaloids are potent
reversible inhibitors of AChE [148].
Huperzine A (39) has been found more potent than huperzine B
(40) in AChE inhibition; however, HupB exhibited a higher thera-
peutic index in comparison with HupA. HupA has been approved in
China as a drug for the treatment of AD (Table 1).
A series of tacrine-huperzine A hybrids also have been synthe-
sized [149]. Huprines Z and Y, (41) with combined carbobicyclic
substructure of ()-huperzine A and the 4-aminoquinoline sub-
structure of tacrine. Huprine Z and huprine Y found to be more
active than both tacrine and ()-huperzine A as inhibitors of both
human and bovine AChE; however they showed intermediate
BuChE inhibitory activity (Table 1) [150,151].
The highest activity was exhibited by compound tacrine and
huprine Y joined with a tether with an N-methylamine group
inserted in the middle of the methylene linker i.e. compound 42
(Table 1).
5.5.1.3. Indole alkaloids. The species Tabernaemontana australis
(Müell. Arg) Miers (sin. Peschiera australis), which flourishes in
South America, has been a source of iboga alkaloids that inhibit
AChE. Four indole alkaloids: coronaridine (43), voacangine (44),
voacangine hydroxyin-dolenine (45) and rupicoline (46), showed
anticholinesteric activity at the same concentration as the reference
compounds physostigmine and galantamine (Table 1) [152].
5.5.1.4. Lycorine alkaloids. Lycorine (47) was the first Amaryllida-
ceous alkaloid with a tetracyclic pyrrole [d,e] phenanthridine
(galanthan) skeleton that demonstrated weak inhibitory activity
against AChE (Table 1). Among more than 20 lycorine related al-
kaloids assoanine (48) displayed the highest AChE activity (Table 1).
Secolycorines prepared from lycorine through chemical modi-
fications also showed inhibitory activity against AChE. Compound
49 with N-methyl and 50 with N-butyl substituent were found to be
more potent than N-ethyl or N-propyl derivatives (Table 1) [153].
5.5.1.5. Isoquinoline alkaloids. Isoquinoline alkaloids represent a
successful template for the identification of potent AChE inhibitors
[154]. One type of structure has been represented by bisbenzyli-
soquinoline (BBIQ) derivatives. Acetylcholinesterase inhibitors
among new synthesized series of isoquinoline and dihy-
droisoquinoline derivatives have been discovered. These
 M. Singh et al. / European Journal of Medicinal Chemistry 70 (2013) 165e188
compounds have been designed as monomeric analogs of BBIQ. 1-
Benzyl-3,4-dihydroisoquinoline and 1-benzyl-isoquinoline struc-
ture displayed AChE inhibitory activity in the micromolar range
[155]. The most active compounds of the series were 51 and 52
(Table 1).
A number of bisbenzylisoquinoline (BBIQ) (53) alkaloids; such
as fangchinoline, atherospermoline, and fenfangjine E, isolated
from root of Stephania tetrandra S. Moore, Menispermaceae family,
were found to inhibit acetylcholinesterase enzyme in the micro-
molar range (Table 1) [156].
Stephaoxocanes belonging to a small family of isoquinoline
alkaloids with a tetracyclic skeleton have been known in folk
medicine for a long time. Several tricyclic analogs of these ste-
phaoxocanidine alkaloids also exhibited AChE inhibitory activity.
Modifications of alkaloid structure included replacement of the
oxocane ring with functionalized alkyl chains. One of the most
potent compounds in this series was found to be the analog of
stephaoxocanidine (54) with a lactone ring i.e. (5,6-dimethoxy-7H-
8-oxa-1-aza-phenalen-9-one), i.e. (55) which exhibited similar
activity to that of Narcissus extracts enriched in galantamine
(Table 1) [157].
5.5.1.6. Xanthostigmine alkaloids and its derivatives
5.5.1.6.1. 2-Arylidenebenzocycloalkanone derivative. The key
feature of these derivatives is a 2-arylidenebenzocycloalkanone
moiety that provides the ability to bind at the AChE peripheral site
responsible for promoting the beta amyloid aggregation. 2-
arylidenebenzocycloalkanone analogs prepared with the aim to
target simultaneously both the catalytic and the peripheral AChE
binding sites. The insertion of an alkoxy spacer chain of suitable
length (three or seven methylene units) and introduction of a ste-
rically encumbering ð-electron-rich arylidene moiety into the
arylidene aryl ring resulted in increased contact area with the PAS.
The selected benzocycloalkanone moieties are benzofuran-3-one,
3,4-dihydro-2H-naphthalen-1-one, chroman-4-one and indan-1-
one [158]. The most active one of the series has been compound
56 (Table 1).
5.5.1.6.2. 3-[X-(Benzylmethylamino) alkoxy] xanthen-9-ones an-
alogs. These new derivatives devoid of the carbamoyl substituent
in the phenyl ring have been synthesized. The effect of the linker
length and the presence of different substituents in the phenyl ring
were both tested [159]. Best results were obtained for the com-
pounds having three (57) and seven (58) methylene units respec-
tively (Table 1).
5.5.1.7. Carbazole alkaloids. Carbazole alkaloids also possess the
acetylcholinesterase inhibitory potential. Mahanimbine (59) an
alkaloid has been isolated from the Murraya koenigii leaves by
solvent extraction, via petroleum ether. Mahanimbine [3, 5-
dimethyl-3-(4-methylpent-3-enyl)-11H-pyrano [5,6-a] carbazole]
contain a carbazole nucleus which is responsible for its activity. The
authors reported the acetylcholinesterase inhibitory activity of
carbazole alkaloids by most widely used method i.e. Ellman’s
method [160].
The various structural feature of carvedilol (60) as inhibitors of
Alzheimer beta-amyloid fibril formation have also been identified.
Based on their work they also studied the beta amyloid inhibitory
potential of various derivatives of carbazole like SB-211475 (61), SB-
209995 (62), 9-acetylcarbazole (63) and hydroxy carbazole (64). All
of the compounds when tested for their beta amyloid fibril for-
mation inhibitory potential have shown the good results [161]
(Table 1).
The berberine derivatives have been synthesized and evaluated
as the potent acetylcholinesterase inhibitors. A simple structuree
activity relationship analysis showed that the AChE inhibitory
181
potency has closely related to the length of the alkylene chain.
Compounds with four methylene groups between the berberine
and aromatic ring units, i.e. with n ¼ 4 being the best inhibitors in
the series. Various derivatives (65, (Table 1)) with good inhibitory
potential have been synthesized with n varying from 2 to 6 [162].
New class of multi target directed ligand for inhibition of
acetylcholinesterase and beta amyloids aggregation in AD has also
been synthesized. Since substituted carbazoles are efficient in-
hibitors of beta amyloids fibril formation, the carbazole moiety of
carvedilol was selected to design a new pharmacophore and have
been joined with the tacrine derivative like 6-cholrotacrine and
synthesized the carbacrine. All the designed compounds were
found to be effective AChEIs in the nanomolar range and more
potent than tacrine and its 6-chloro derivative. In particular, car-
bacrine was able to inhibit AChE activity in the nanomolar range,
block in vitro beta amyloids self aggregation and aggregation
mediated by AChE, antagonize NMDARs and reduce oxidative
stress, and the most active compound of the carbacrine series was
compound (66) (Table 1) [163].
5.5.2. Terpenoids
Novel meroterpenoid AChE inhibitors have been isolated from
microbial metabolites. Solid state fermentation of Aspergillus ter-
reus led to isolation of terreulactones AeD and another polar
metabolite isoterreulactone-A (67). Terreulactone A (68) and Ter-
reulactone D (69) are meroterpenoid type compounds; with mixed
polyketide terpenoid structures that showed AChE inhibition
(Table 1). Isoterreulactone A is also a meroterpenoid but contains a
seven-membered lactone skeleton in its structure and it is 10 times
weaker AChEI than terreulactones [164,165].
These new inhibitors (70, (Table 1)) contain a modified het-
erocycle which is supposed to interact with the catalytic site and
new substituents connected to the nitrogen atom N 20, which is
supposed to be responsible for the binding to the peripheral site of
the enzyme [166].
5.5.3. Steroids
Natural cholinesterase-inhibiting steroids isolated from Sarco-
cocca saligna have been reported. These pregnane-type compounds
with steroidal skeleton and monomethylamino or dimethylamino
substituents, either at C-3 and/or at C-20 position of the basic
steroidal skeleton have been found to inhibit both AChE and BuChE
in micromolar ranges. Among over twenty new steroids, the most
active ones were salignenamide-E (71), salignenamide-F (72) and
axillaridine-A (73, (Table 1)). All investigated steroids contain
amino nitrogen atoms at positions C-3 and/or C-20. These play an
important role in the inhibitory activity of these compounds
[167,168].
5.6. Miscellaneous AChE inhibitors
5.6.1. Aminobenzoic acid derivatives
The arylamides and arylimides structurally related with ACh,
derived from the p-aminobenzoic acid (PABA) have been designed
and developed [169]. Various meta and para aminobenzoic acid
derivatives act as the potential AChE inhibitors [170,171]. Among
the tested PABA derivatives, the most active have been compound
74 and 75 (Table 1) [170,172].
5.6.2. 1-[Bis(4-fluorophenyl)-methyl] piperazine derivative
Several derivatives of 1-[bis(4-fluorophenyl)-methyl] pipera-
zine with various heterocyclic rings have been synthesized that
exhibited AChE inhibitory activity [171].The highest potency
against AChE was exhibited by compound (76, (Table 1)) with
pyrrolidine-substituted piperazine.
182 M. Singh et al. / European Journal of Medicinal Chemistry 70 (2013) 165e188
5.6.3. Obidoxime and structurally related oximes
Several analogs of obidoxime, derivatives of pyridinium oxime
ether have been developed. It has been found that the monopyr-
idinium compound and the bispyridinum compound displayed
inhibitory activity towards AChE in the low micromolar range. The
greatest inhibitory activity observed for compound 77 with mono
chloro and 78 with 2,6-dichloro substituents in the phenyl ring
(Table 1). These dimeric compounds may interact with both active
and peripheral binding sites of the enzyme [173e175].
5.6.4. Thienoxazinones derivatives
There have been several 2-secondary-amino-substituted thie-
noxazinones derivatives compound 80, with inhibitory activity
against AChE (Table 1). It contains a bulky benzyl residue attached
to the basic nitrogen atom. Moreover, 2-secondary-amino-
substituted thienoxazinones led to discovery of new
tetrahydropyrido-anellated thieno [1,3]-oxazinones as potent in-
hibitors of AChE. Among the tricyclic 1,3-oxazin-4-ones, com-
pounds 79, 80, 81 and 82 showed inhibitory activity in the sub
micromolar range (Table 1) [176].Thus, these compounds may bind
to the active site gorge of AChE in a manner similar to donepezil.
5.6.5. Phenothiazine derivatives
Various derivatives of phenothiazine (83, (Table 1)) have been
synthesized. Some of the synthesized derivatives inhibited both
BuChE and AChE [177].
5.6.6. Quinazolinimines
There have been potent inhibitors of cholinesterases in the se-
ries of Quinazolinimines [178].Among new synthesized molecules,
compounds 84e87 showed moderate inhibition activity against
cholinesterase (Table 1). Novel tricyclic Quinazolinimines, tetracy-
clic dibenzodiazocines and related analogs have also been synthe-
sized and tested [179].
5.6.7. Bis-()-nor-meptazinol derivative
A bis-()-nor-meptazinol derivative (88), in which the two
meptazinol rings being linked by a nonamethylene spacer, a novel
acetylcholinesterase inhibitor inhibits both catalytic activity and
beta amyloid peptide aggregation (Table 1) [180].
5.6.8. Cis-2,6-dimethyl piperidine sulphonamides derivative
Donepezil is a widely prescribed AChE inhibitor, which displays
a piperidine ring in its structure. These piperidine sulfonamides
have been subjected to in vitro AChE enzyme inhibition studies
[181].The most active compound of series showing the AChE
inhibitory potential were 89 and 90 (Table 1).
5.6.9. Carbamate derivative
5.6.9.1. Substituted phenyl-N-butyl carbamates. These compounds
(91, (Table 1)) have been found to possess potent, irreversible, pe-
ripheral anionic site-directed inhibition of AChE [182]. The X can be
substituted with H to OMe, NO2.
5.6.9.2. Alkane-1-N-butylcarbamate-n-ols and 1,n-alkane-di-N-
butylcarbamates. These compounds are identified as “pseudo-
irreversible” (“pseudo-substrate”) inhibitors of AChE. The most
active one has been the hexadecane derivative 92 with one car-
bamoyl group (Table 1). The authors also suggested that com-
pounds, which contained two carbamate moieties, interacted with
both peripheral and catalytic active sites [183].
5.6.9.3. Phenylcarbamates. Phenylcarbamates structurally related
to Rivastigmine were evaluated, in vitro and in vivo for biological
activity. Among these compounds that showed the highest activity
is 1-[1-(3-dimethylcarbamoyloxyphenyl) ethyl] piperidine] (93,
(Table 1)). Meta-substituted derivatives inhibited cholinesterases
more strongly than ortho-substituted compounds [184].
5.6.10. 2-Phenoxy-indan-1-one derivatives
These derivatives comprise a new group of cholinesterase in-
hibitors with a dimeric structure i.e. two pharmacophoric moieties
i.e. 5, 6-dimethoxy-indan-1-one derived from Donepezil and
dialkyl-benzylamine derived from Rivastigmine. These compounds
are able to interact with central and peripheral binding site of AChE
and prevented catalytic and noncatalytic actions of the enzyme
[185].The compounds 94 and 95 exhibited the highest activity
among all of the designed compounds (Table 1).
5.6.11. Nelumbo nucifera
N. nucifera is an aquatic plant with numerous medicinal prop-
erties. The primary effect of this plant is as an AChE inhibitor rather
than as BACE1 inhibitors [186].There are no reports of human
studies. Preclinical and clinical safety and toxicity data is not
reported.
5.6.12. Himatanthus lancifolius
H. lancifolius is a shrub. Seidl et al. conducted a study to deter-
mine if there were any AChE inhibiting properties from the H.
lancifolius extract (Seidl et al., 2010). The dichloromethane (DCM),
and ethyl acetate (EtOAc), fractions showed significant AChE
inhibitory effects and Uliene was the significant compound present
in both fractions [187].
5.6.13. Galangin
Guo et al. studied 21 different flavonoids for potential AChE
inhibition properties in the brain in vitro. Flavonoids have been of
great interest in AD research and treatment because of their free
radical scavenging properties. A flavonol isolated from Rhizoma
Alpiniae officinarum called galangin demonstrated the highest
inhibitory effects on AChE activity [188].Clinical and preclinical
toxicities have not been established yet.
5.6.14. Cardanol derivatives
De Paula et al. designed new AChEI from nonisoprenoid
phenolic lipids (NIPLs) of Anacardium occidentale. The study
concluded that the most promising candidates to the development
of AchEI for AD treatment were derived from cardanol. Clinical and
preclinical toxicities have not been established yet [189].
5.6.15. Metrifonate
Metrifonate is a long-acting irreversible ChEI that was originally
used to treat schistosomiasis. The clinical trials were discontinued
during phase III. Metrifonate is not an option for AD treatment at
this time. Metrifonate is not an approved AD treatment but showed
efficacy that was outweighed by safety risks [190].
5.6.16. Coumarin derivatives
Coumarins are naturally occurring phytochemicals in many
plant species. Coumarins primarily interact with PAS of AChE, so
scientists have put their efforts in synthesizing dual inhibitors of
AChE by incorporating a catalytic site interacting moiety with
coumarin through an appropriate spacer. Coumarin derivatives are
recently reviewed by Anand et al. [191].
5.6.17. Oxoisoaporphine-based inhibitors
A series of novel oxoisoaporphine-based inhibitors (10-
aminoalkylamino-1-azabenzanthrone AreNH(CH2)nNR1R2) of
acetylcholinesterase (AChE) has been designed, synthesized, and
tested for their ability to inhibit AChE, butyrylcholinesterase (BChE)
 M. Singh et al. / European Journal of Medicinal Chemistry 70 (2013) 165e188
and AChE-induced b-amyloid (Ab) aggregation. Molecular docking
simulations on the oxoisoaporphine derivatives with AChE from T.
californica have demonstrated that 1-azabenzanthrone moiety of
the ligands could interact with peripheral anionic site (PAS) of
acetylcholinesterase, especially with Trp 279 of PAS. A series of
oxoisoaporphine derivatives 96 with different basic side chain
(n ¼ 2 and 3) at 10-position of 1-azabenzanthrone were designed
and synthesized, and their anti-AChE, BChE and AChE-induced
Ab140 aggregation activities were tested [192].
The structure of terminal groups of side chain has effect on their
inhibitory activities. High inhibitory potency was found to be
associated with diethylamine at the end of side chain whereas
dimethylamine, pyrrolidine and piperidine derivatives showed less
potency. Compounds which possessed hydroxyl group at the end of
side chain exhibited much weaker anti-AChE potency, which
caused approximately 16- to 340-fold decrease compared with
diethylamine derivatives in activity. Compound 102 (IC50 of
0.72  0.03 uM) showed the highest inhibitory activity against
AChE and BChE (13.4 mM) (Table 1). The anti-Ab aggregating effect
seems to be dependent on the length of the side chain. Since the
inhibitory potency was increased by increasing the length between
the 1-azabenzanthrone and terminal nitrogen atoms.
5.6.18. Tricyclic analogs of stephaoxocanidine
The synthesis of simplified analogs of the novel isoquinoline
alkaloid stephaoxocanidine, carrying the oxazaphenalene ABC-ring
system of the natural product, and their activity as inhibitors of the
enzyme acetylcholinesterase, is reported [157]. 5,6-Dimethoxy-7H-
8-oxa-1-aza-phenalen-9-one (97, (Table 1)) was as active as a
Narcissus extract enriched in galantamine. The stephaoxocanes are
a small family of isoquinoline alkaloids recently uncovered by
Japanese, Chinese and Brazilian scientists, which shared the tetra-
cyclic skeleton 98 (Table 1). Till date, only five members are known
i.e. stephaoxocanidine (99) and stephaoxocanine (100, (Table 1))
isolated from Stephania cepharantha Hayata, excentricine (101,
(Table 1)) and N-methylexcentricine (102), from Stephania
excentrica and eletefine (103, (Table 1)) isolated from Cissampelos
pareira [193e195].
Interestingly, besides galantamine other alkaloids such as
isoquinoline derivatives from Amarillidaceae as well as
Fig. 10. Tacrine-4-oxo-4H-chromene hybrids.
183
protoberberines, and quaternary benzophenanthridine and iso-
quinoline alkaloids including sanguinarine and N-alkyl carnegi-
nium salts, have been shown to display acetylcholinesterase
inhibitory activity [196e198]. It was observed that lactone exhibi-
ted an IC50 of 19.6 mM, a remarkable activity.
Seven ABC-ring analogs of stephaoxocanidine have been syn-
thesized and their activity as inhibitors of acetylcholinesterase was
tested. Lactone 100 (Table 1), was found to be the most potent
compound of this series. Transformation of the lactone moiety
furnished less active compounds but did not abolish the acetyl-
cholinesterase inhibiting activity. Unexpectedly, however, intro-
duction of a functionalized side chain partially resembling ring D of
the tetracyclic natural products, did not improve the activity.
5.6.19. Oxoisoaporphine and oxoaporphine derivatives
Oxoaporphine (104, (Table 1)) alkaloids were designed and
synthesized as acetylcholinesterase (AChE) and/or butyr-
ylcholinesterase (BuChE) inhibitors [192]. The AChE inhibitory po-
tential of synthetic oxoaporphine derivatives was decreased about
2e3 orders of magnitude as compared with that of oxoisoapor-
phine derivatives. The synthetic oxoisoaporphine derivatives
exhibited high AChE inhibitory activity with IC50 values in the
nanomolar range and high selectivity for AChE over BuChE (45- to
1980-fold).
Newly synthesized oxoisoaporphine derivatives, 9-(3-
piperidinopropionamido)-1-azabenzanthrone methiodide salt,
prospectively showed the most powerful inhibitory potency toward
AChE with IC50 value in sub-nanomolar level. The only difference
between oxoaporphine (105, (Table 1)) and oxoisoaporphine (106,
(Table 1)) alkaloids is the position of nitrogen atom in the
pharmacophore.
5.6.19.1. Hybrids of oxoisoaporphine-tacrine congeners. The new
hybrids consist of a unit of 1-azabenzanthrone and a tacrine (107,
(Table 1)) or its congener, connected through an oligomethylene
linker containing an amine group at variable positions [199]. These
hybrids exhibit high AChE inhibitory activity with IC50 values in the
nanomolar range in most cases. Those, bearing a tetrahydroacridine
moiety, exhibit a significant in vitro inhibitory activity toward the
AChE-induced and self-induced Ab aggregation, which makes them
promising anti-Alzheimer drug candidates.
It was predicted that hybrids of oxoisoaporphine-tacrine con-
geners in which the two pharmacophores were separated by a
linker of a suitable length would have both greater inhibitory po-
tency and selectivity than tacrine or oxoisoaporphine itself and
should be involved in neurotrophic activity. These compounds
consist of a unit of tacrine or its congeners, which occupies the
same position as tacrine at the AChE active site, and the 1-
azabenzanthrone moiety whose position along the enzyme gorge
and the peripheral site can be modulated by a suitable tether that
connects tacrine and 1-azabenzanthrone. The proper tether length
for the linker between the two anchoring groups, 9-aminoacridine
and 9-amino-1-azabenzanthrone, seemed to be six. Various fla-
vones were used as adjuvant with some active moieties due to their
antioxidant activity. In that context, a new family of tacrine-4-oxo-
4H-chromene hybrids has been designed, synthesized, and evalu-
ated biologically for Alzheimer’s disease (AD) [200] (Fig. 10).
5.6.20. Recent synthetic analogs
The first AChEI developed was THA, but using the drug caused
dose-dependent but reversible liver toxicity. The 7-methoxytacrine
(7-MEOTA) is an analog of THA that has far less toxicity and is
pharmacologically equivalent to THA [201]. In a study by Kor-
abecny, fourteen 7-MEOTA analogs were synthesized. Models of
human recombinant AChE (hAChE) and human plasmatic BuChE
184 M. Singh et al. / European Journal of Medicinal Chemistry 70 (2013) 165e188
(hBuChE) were used to evaluate these new analogs in vitro and
were compared to THA and 7-MEOTA. Ladostigil is a novel anti-
Alzheimer’s disease drug with neuroprotective, multimodal brain-
selective monoamine oxidase, and cholinesterase inhibitor prop-
erties [202]. Ladostigil (108, (Table 1)) also prevented the age-
related reduction in cortical AChE activity and the increase in
butyrylcholinesterase activity in the hippocampus presently in a
Phase II clinical trial and intended for the treatment of Alzheimer’s
disease and dementia co morbid with extrapyramidal disorders
and depression.
AP2238 (109, (Table 1)) is the first published compound to bind
both anionic sites of AChE. The potency against AChE is comparable
to donepezil, while its ability to contrast b-amyloid aggregation is
higher. There are reports on a series of hybrids developed from
donepezil and AP2238 in which the indanone core from donepezil
is linked to the phenyl-N-methylbenzylamino moiety from AP2238.
There are no reports of human studies. Preclinical and clinical
safety and toxicity have not been established yet [203].
A great deal has been learned about the pathogenesis of neu-
rodegeneration, after less than three decades. Novel intervention
strategies are being developed to improve the neuro-toxicity
caused by abnormal metabolic products and prevent processes
that lead to cell death. A large number of clinical trials are under-
way, both industry and government sponsored studies with widely
used drugs (e.g. antioxidants, anti-inflammatory agents, statins and
vitamins) that might reduce the risk of Alzheimer’s disease.
Intensive studies are underway on multiple fronts, from basic sci-
ence to genetics to drug therapy to care giving. Some of the key
factors that influenced the pace of progress and helped to change
the ‘status’ of dementia research were: a) increases in research
funding, b) recruitment of new scientific talent; convergence of
know-how and technologies, c) several crucial discoveries in mo-
lecular neurobiology.
6. Conclusion
Research is always driven by the funding, which in turn gener-
ates facilities. Drug discovery is a tedious process and involves a lot
of time as well as money. Research funding has always been to
achieve a myopic goal. The path of research on AD started in 1906,
but the research was hampered by the war times. In the post war
time, as the population grew older the research was reoriented
towards the study of Alzheimer. The path of research of AChE in-
hibitors started in 1876, with use of these inhibitors in glaucoma.
Then the toxicity of these compounds was misused for pest man-
agement and pesticides came into existence. With the eruption of
war, toxic compounds of this category became more toxic nerve
agent. In this era all, the funding was going in the development of
nerve gases. Their mechanistic studies were studied in detail to
counter adverse actions and antidotes came into the picture. Again,
after the war, funding went for wellbeing of younger population for
development of antibiotics and immunization. As the population
grew older, AD again came to front row of funding. These two paths
merged with discovery of the fact that brain Acetylcholine levels
are depleted in AD. Normally, identification and validation of target
is a time consuming process. The whole research time was reduced
with development of AChE inhibitors as drug for AD due to highly
developed research facilities in the field of AChE during the world
war era. Hence, the first four drugs that were approved for AD
belonged to this class. As it is a past therapy of AD, numerous new
approaches such as Tau-based therapies, dealing with oxidative
stress, targeting cellular Ca2þ handling, anti-inflammatory therapy,
amyloid targeted strategies (b-Secretase inhibitors and g-Secrea-
tase modulators) are currently being employed for the treatment of
AD. Among them, b-Secretase (BACE) inhibitors and g-Secreatase
modulators (GSMs) are emerging as promising AD therapy. In
recent times, many companies and academic groups have ongoing
b-Secretase inhibitors and GSMs development programs and also
some of them have entered early phase clinical trials but are still
lagging behind in the studies. On the other hand, the potential of
AChE inhibitors is well explored and hence it won in the race of
drug development in AD treatment.
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