Advances in drug discovery to assess cholinergic neurotransmission: a systematic review.

Abstract

Neurotransmission is essential to physiological processes of cellular communication. The search for new molecules that may influence neurotransmission systems is an open field with possible impact on several pathophysiological conditions or diseases: Alzheimer's disease, Parkinsonism and myasthenia gravis, etc. The present review describes the most important aspects of cholinergic neurotransmission, as well as natural and synthetic compounds that, as clinical or experimental drugs, are able to influence this transmission. The pharmacological effects of substances that bind to muscarinic or nicotinic cholinergic receptors, along with their corresponding affinities will also be presented.

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Current Drug Discovery Technologies, 2008, 5, 000-000
1
1570-1638/08 $55.00+.00 © 2008 Bentham Science Publishers Ltd.
Advances in Drug Discovery to Assess Cholinergic Neurotransmission: A Systematic
Review
Vitor F. Ferreira*,1, David R. da Rocha1, Kátia G. Lima Araújo2 and Wilson C. Santos3
1Departamento de Química Orgânica, Instituto de Química, Universidade Federal Fluminense, Outeiro de S. João Batista s/n Centro
24020-150, Niterói, RJ, Brazil
2Departamento de Bromatologia, Faculdade de Farmácia, Universidade Federal Fluminense, Rua Mário Viana 523, Santa Rosa
24241-000, Niterói, RJ, Brazil
3Departamento de Farmácia e Administração Farmacêutica, Faculdade de Farmácia, Universidade Federal Fluminense, Rua Mário
Viana 523, Santa Rosa 24241-000, Niterói, RJ, Brazil
Abstract: Neurotransmission is essential to physiological processes of cellular communication. The search for new molecules that may
influence neurotransmission systems is an open field with possible impact on several pathophysiological conditions or diseases: Alz-
heimer’s disease, Parkinsonism and myasthenia gravis, etc. The present review describes the most important aspects of cholinergic neu-
rotransmission, as well as natural and synthetic compounds that, as clinical or experimental drugs, are able to influence this transmission.
The pharmacological effects of substances that bind to muscarinic or nicotinic cholinergic receptors, along with their corresponding af-
finities will also be presented.
Key Words: Cholinergic neurotransmission, cholinergic receptors, carbohydrates.
INTRODUCTION
Since the beginnings of mankind, much effort has been devoted
to comprehending human bodily functions. At the beggining, the
search for information about ourselves was basically driven by curi-
osity. However, over the centuries, sanitary, social and economical
aspects have become the concerns driving research.
Nevertheless, and despite the great advances, understanding of
how the nervous system works is still a challenge. For example, the
morphophysiopathological changes in cholinergic neurotransmission,
an essential physiological event that controls a variety of human func-
tions and may determine many disorders and diseases, still need to be
better understood. Continued efforts to study the mechanisms control-
ling these biological functions and to speed up the development of
new drugs with high degree of specificity and fewer side effects are
necessary. New drugs are needed to treat some still not understood
diseases. Thus, Parkinson’s [1] disease, Alzheimer’s [2] disease, mus-
cular dystrophies, motor neuron diseases and myasthenia gravis [3],
are all disorders in which new therapeutic approaches are urgently
needed. Nowadays the pharmacologic approaches to these diseases
are mostly palliative and it is not easy to prevent their progression.
Drugs that will improve quality of life as well as reduce their serious
impact on public health will always be desirable.
In this review we wish to present an overview of the currently
understood biochemical aspects of cholinergic neurotransmission and
different synthetic and natural molecules with a view to considering
their usefulness in some diseases involving cholinergic neurotrans-
mission.
NEUROTRANSMISSION
Neurotransmission is the process whereby nervous system cells
communicate among themselves and with others cells of the organ-
ism through nerve impulses. This process produces responses in
smooth, cardiac and skeletal muscles and glands [4]. This fundamen-
tal physiological event is mediated by some specific chemical com-
pounds called neurotransmitters. Many of them are involved in di-
verse body functions: they include noradrenaline, dopamine and


*Address correspondence to this author at the Departamento de Química Orgânica, Insti-
tuto de Química, Universidade Federal Fluminense, Outeiro de S. João Batista s/n Centro
24020-150, Niterói, RJ, Brazil; E-mail: cegvito@vm.uff.br
histamine [4]. Acetylcholine (ACh, 1, Fig. (1)), the first neurotrans-
mitter to be identified, acts in the central and peripheral cholinergic
nerves of many organisms [5] and is involved in many functions,
including excitability [6], attention [7, 8], learning [9], memory [10,
11] and stress response [12].




Acetylcholine (1)
Fig. (1). Acetylcholine (1) chemical structure.
Acetylcholine (1) is biosynthesized in specific neurons by choline
acetyltransferase, which uses choline and acetyl coenzyme A as pre-
cursors. The resulting neurotransmitter is stored in synaptic vesicles,
from where it is released to the synaptic cleft by depolarization in a
process called exocytosis [4]. The enzyme acetylcholinesterase
(AChE) converts acetylcholine into the inactive metabolites choline
and acetate [13, 14]. AChE is abundant in the synaptic cleft, and its
role in rapidly clearing free acetylcholine from the synapse is essen-
tial for proper muscle function [4]. Once released, the neurotransmit-
ter (1) acts as the physiologic ligand on the pre- and post-synaptic
receptor sites [15] (Fig. (2)).
In the synaptic cleft, acetylcholine binds to two kinds of choliner-
gic receptors: a) muscarinic receptors (M), which belong to the me-
tabotropic receptor family; and b) nicotinic receptors (N), which be-
long to the ionotropic receptor family [16]. This nomenclature origi-
nated in the observation that acetylcholine mimicked the pharmacol-
ogical effects of the natural alkaloids, muscarine (2) and nicotine (3),
that were employed to discover the receptors (Fig. (3)).
NICOTINIC RECEPTORS
Nicotinic acetylcholine receptors (nAChRs) are proteins with a
molecular weight of about 280 kDa that are found in the central and
peripheral nervous system [17]. These receptors are made up of five
subunits (?, ?, ?, ? or ?) that combine to form homologous or identical
pentamers, and are arranged so as to form a central ionic channel [18-
21] (Fig. (4)). The nAChRs muscle form is located at the final portion
of the neuromuscular junction, and has five subunits: two ? subunits,
one ? subunit, one ? subunit and either a ? or a ? subunit, while the
neuronal forms are much more heterogeneous, presenting a wide
MeO
O
N
Me
Me
Me
Cl

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2 Current Drug Discovery Technologies, 2008, Vol. 5, No. 3
Ferreira et al.
range of possible subunit combinations. Until now, ten types of
subunits ? and four ? nicotinic receptors have been described as com-
posing the receptor sites [18, 22]. Depending on their subunit combi-
nation, nicotinic receptors show different activation and inactivation
kinetics and variable physiopharmacological properties [14].





Muscarine (2)
Fig. (3). Alkaloids muscarine (2) and nicotine (3).
This diverse composition suggests the existence of a large num-
ber of nicotinic receptors [23], and makes it difficult to study and
characterize them [24]. Since nAChRs are widely distributed in the
body and are involved in fundamental physiopharmacological func-
tions [25] interest has grown both in their involvement in and in their
possible usefulness as therapeutic targets [26]. In fact, nicotine (3),
the archetype nicotinic receptor ligand, is responsible for a variety of
central and peripheral pharmacological effects on cardiovascular,
gastrointestinal and endocrine activities that affect cognitive func-
tions, such as analgesia, neurotransmitter release and neuroprotection
[27].
As mentioned before, nAChRs belong to the ionotropic receptor
family [16], which form ligand gated ion channels in cellular plasma
membranes. All nAChRs are permeable to Na+ and K+, and some
subunit combinations are also permeable to Ca2+. The opening of the
channel allows positively charged ions, in particular, sodium and
calcium, to enter in and depolarize the cell [28].
The nAChRs are involved in several nervous system disorders
[4]. Understanding the interaction of natural and synthetic substances
with these receptors is important for medical science [29]. For in-
stance, epibatidine (4) and dimethyl-4-phenyl-piperazine (DMPP) (5,
Fig. (5)) are nicotinic receptor agonists. They mimic the acetylcholine
response when bound to the receptors and have been employed in
many experiments examining the complex functions and structure of
nicotinic receptors in the hope of delimiting the possible role of these
receptors in neurodegenerative diseases [30].
Nicotinic receptor antagonists (Fig. (6)), for example, pancu-
ronium (6) and rocuronium (7), inhibit ACh binding to receptors [30].
These substances, and other analogues, are widely employed as neu-
romuscular in anesthesia. Neuromuscular blocking drugs relax the
skeletal muscles and induce paralysis [4]. Recently, experiments us-
ing bovine adrenal cells as experimental model evidenced the direct
action of atropine (8) on nicotinic receptors at nanomolar level con-
centrations: atropine blockaded of catecholamine release [31]. This
observation is the opposite of the classic belief that atropine (8)
blocks nicotinic cholinergic receptors only in very specific situations













Fig. (2). Schematic illustration of cholinergic neurotransmission.









Fig. (4). Schematic illustration of nicotinic receptor.
O
OH
Me
N
Me
Me
Me
N
N
Me
Cl
Nicotine (3)

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Advances in Drug Discovery to Assess Cholinergic Neurotransmission Current Drug Discovery Technologies, 2008, Vol. 5, No. 3 3
and at a milimolar concentration [32-34]. This finding also indicates a
new role for the therapeutic and experimental uses of atropine (8), as
its use at a nanomolar concentration can selectively block cholinergic
functions where neurotransmission is controlled by muscarinic and
nicotinic receptors.





Fig. (5). Examples of nicotinic receptors agonists.
MUSCARINIC RECEPTORS
In 1914, Sir H. Dale, in a classic paper on the chemistry and
pharmacology of cholinergic muscarinic receptors [16] first described
the use of muscarine (2) as a selective agonist and atropine (8) as a
selective antagonist of these receptors. Since then, advances in
physiopharmacology and molecular biology, have led to a better un-
derstanding of these receptors [4].
Muscarinic receptors are part of the metabotropic receptor family,
in which receptors are coupled on their internal face to G proteins
(Fig. (7)). The receptor is formed by a single glycoprotein with seven
transmembrane loops. The extracellular amine termination (NH2)
possesses glycosylation sites and a carboxylic termination. The site
for acetylcholine binding on the receptor is not yet clearly identified
[35, 36]. Five genes codifying the proteins that compose the mus-
carinic receptors (M), have already been cloned and characterised
[37-39]. Five kinds of muscarinic receptors, named from M1 to M5,
based on their pharmacologic specificity have been generated [40].
Each receptor subtype is related to different G proteins that can be
modulated in their own or by a second messenger, thus activating the
ionic channel.
The muscarinic receptors (M) are involved in the regulation of
smooth muscle contraction, gland secretion, cardiac rate and have
effects on the central nervous system, on motor control, temperature
regulation and cognitive functions. Smooth muscle contraction, glan-
dular secretion, pupil dilatation and food intake are mainly mediated
by M3 receptors, whilst the effects on cardiac rate are mainly medi-
ated by M2 subtypes [41, 42].
















Fig. (6). Examples of nicotinic receptor antagonists.















Fig. (7). Schematic illustration of muscarinic receptor.
H
N
NCl
NN
Me
Me
I
DMPP (5)
(-)-Epibatidine (4)
NMe
O
O
OH
H
HO
N
Me
Me
O
N
H
H
O
Me
O
H
H
Me
O
O
O
Me
Me
H
H
H
Me
H
N
O Me
N
Me
Br
Br
Br
Rocuronium (7)
Pancuronium (6)
Atropine (8)

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4 Current Drug Discovery Technologies, 2008, Vol. 5, No. 3
Ferreira et al.
Muscarinic receptor agonists, like pilocarpine (9) and (S)-
bethanechol (10) (Fig. (8)), mimic acetylcholine action [30]. Among
other effects, they increase secretion of exocrine glands, reduce blood
pressure, and increase intestinal motility and ocular pressure. This
makes, analogues of this class of compounds very interesting because
the possibility of their applicability in the treatment of diseases like
glaucoma and Alzheimer’s disease [43].






Fig. (8). Examples of muscarinic receptor agonists.
Muscarinic receptor antagonists like tropicamide (11) and (R)-
procyclidine (12, Fig. (9)), inhibit acetylcholine effects by binding to
receptors on cardiac and smooth muscle, glands, peripheral ganglions
and central nervous system cells [30]. Additionally, these compounds
have been used, or are of therapeutic interest, in other conditions
including asthma, glaucoma, Parkinson’s disease, intestinal motility,
cardiac and bladder dysfunctions [44].







Fig. (9). Examples of muscarinic receptors antagonists.
As previously mentioned, muscarinic receptors (1) play a funda-
mental role in the central and peripheral nervous system. Several
recent studies have been devoted to preparing new compounds with
agonist and antagonist activities, and higher specificity and selectivity
to each receptor subtype [45].
SUBSTANCES WITH ACTIVITY ON CHOLINERGIC
NEUROTRANSMISSION
Given the great variety of nicotinic and muscarinic receptor sub-
types, the synthesis of substances that selectively bind to these recep-
tors is a challenge. In order to find new potential candidate drugs to
act on cholinergic neurotransmission, natural products have been an
inspiration for organic synthetic chemists, who have produced many
prototypes of substance analogues for these models [46].
NICOTINIC RECEPTORS AGONISTS AND ANTAGONISTS
This section will describe examples of substances with a nicotinic
receptor agonist and/or antagonist activity and that have a natural or
synthetic origin. Selectivity for nicotinic receptors subtypes ?4?2, ?3?4
and ?7, is expressed as Ki (affinity constant) and classified as: high,
when Ki < 1 nM; modest, when 1 ? Ki ? 10 nM and low when Ki >
10 nM. Interestingly the ?4?2 and ?7 receptors subtypes are found in
the central nervous system while the ?3?4 subtype is found in gan-
glious nervous system [47].
Acetylcholine Analogs (1)
The structure of some tertiary and quaternary amine analogues of
acetylcholine (1), choline (13) and DMPP (5) are shown in Table 1.
Choline (13) has low affinity for nicotine receptors, partially ex-
plained by its fast desensitisation. Carbamylcholine (14) differs from
choline in that its alcohol function is transformed into carbamate, thus
increasing its affinity for nicotinic receptor subtypes, particularly the
?4?2 receptors. The transformation of the carbamylcholine carbamate
into N-methylcarbamate (e.g., 15) strong increases the affinity of this
substance for ?4?2 receptor subtypes. The nicotinic receptor affinity
of DMPP (5) is low, but higher than that of N-methylcarbamate
probably because of the rigid structure of 15. On the other hand, the 5
analogue which has a larger heterocyclic ring, e.g. 16, also shows
high affinity for ?4?2 receptors.
Nicotine (3) Analogs
Many synthetic compounds that are structurally related to nico-
tine (3) have been described in the literature. Some of them are de-
scribed in Table 2. Nicotine (3) is a natural alkaloid found in many
plants. The compound shows modest affinity for the ?4?2 receptor
subtype. The introduction of a chlorine atom in the pyridinic ring, e.g.
6-chloronicotine (17), improves its affinity for this receptor subtype.
Substitution of the nicotine (3) pyrrolidinic ring with an aliphatic
amine with same the molecular formula reduces affinity for the three
nicotinic receptor subtypes, as occurs with metanicotine (18). The N-
methyl group is critical to the activity of 3. Substituting this group
with a propyl group, e.g. 19, dramatically reduces the affinity for the
receptor site and converts it into a nicotinic antagonist. The same
trend is observed when a pyrrolidinic ring is substituted by a quinu-
clidinic ring, but this does not change the affinity for the ?4?2 receptor
subtype as compared to compound 20.
TROPANIC ALKALOIDS ANALOGS
A potent neurotoxin produced by the cyanobacterium Anabaena
flos-aquae (21, Table 3), (+)-anatoxine-a causes paralyses respiration
in rats (LD50 = 0.2 mg/Kg) [48]. This substance was first described in
1977 and was demonstrated to have high affinity and selectivity for
?4?2 receptors [49]. The replacement of the hydrogen atom in the
nitrogen is very important for its activity, and N-methylation of 21
reduces its activity, as observed for 22. Transformation of the acetyl
group in 21 into a methyl ester (e.g. 23) decreases the activity of the
compound. The natural alkaloid (+)-ferruginina (24), isolated from
the arboreal species Darlingia ferruginea [50] and D. darlingiana
[51], has a smaller bicyclic ring than (+)-anatoxine-a. This structural
modification dramatically decreases its affinity for the nicotinic re-
ceptors.
Anabaseine (25) Analogs
Anabaseine (25, Table 4)
worms, that was subsequently also found in certain ants species [52,
53]. Its structure is very similar to that of nicotine (3) [54] and it has a
moderate affinity for nicotinic receptors, along with its analog with an
expanded ring, 26. Other analogues of 25, such as compounds 27 and
28, are antagonists for the ?4?2 receptor, despite being potent receptor
?7agonists.
is a natural toxin produced by marine
Epibatidine Analogs (4)
The discovery of epibatidine (4, Table 5) by Daly and coworkers
[55], in the skin of the Ecuadorian frog Epipedobates tricolor had a
tremendous impact on nicotinic receptor research, given its high af-
finity for ?4?2 e ?3?4 receptors [56] (Table 5).
The substitution of the 6-chloro-pyridine group in epibatidine (4)
with 3-methyl-isoxazol converted it into epiboxidine (29) which had
reduced receptor affinity. Similar reductions in affinity were also
observed when the bicycle ring in 30 was expanded.
PYRIDILETHERS
The pyridilethers are nicotine (3) analogs that act on nicotinic re-
ceptors. For instance, pyridilether 31 (Table 6) shows high affinity for
the ?4?2 nicotinic receptors. Demethylating the pyrrolidine ring on the
O
O
Me
N
N
Me
N
O
Me
Me
Me
Me
O
NH2
Cl
Pilocarpine (9)
(S) -Bethanechol (10)
H
N
OH
Cl-
N
NMe
O
OH
(±)Tropicamide (11)
(R)-Procyclidine (12)

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Advances in Drug Discovery to Assess Cholinergic Neurotransmission Current Drug Discovery Technologies, 2008, Vol. 5, No. 3 5
Table 1.

Selected Agonists Structurally Related Acetylcholine (1) and their Action on the Nicotinic Receptors [47]
Ki (nM)
Substance
?4?2
?3?4
?7
Source
N
OHMe
Me
Me
Cl
Choline (13)

7,000 - 180,000 Natural
N
OMe
Me
Me
NH2
O
Cl
Carbamylcholine (14)

61 1,300 22,000 Synthetic
N
O
H
NMe
Me
Me
O
Me
Cl
N-Methylcarbamylcholine (15)

2.0 460 11,000 Synthetic
NN
Me
Me
I
DMPP (5)

18 220 7,600 Synthetic
HN
N
N
Cl
OCH3
(16)

0.00068 - - Synthetic

Table 2.

Selected Agonists and Antagonists Structurally Related to 3
Ki (nM)
Substance
?4?2
?3?4
?7
Source
N
N
Me
Nicotine (3)

1,0 73 1,600 Natural
N
Me
6-Chloronicotine (17)
N
Cl

0.63 - - Synthetic
N
N
H
Me
Metanicotine (18)

24 - 36,000 Synthetic
N
N
Me
(19)

22 * - - Synthetic
N
N

* Antagonist
1.0 * - - Synthetic

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6 Current Drug Discovery Technologies, 2008, Vol. 5, No. 3
Ferreira et al.
Table 3.

Selected Agonists Structurally Related to Tropanic Alkaloids
Ki (nM)
Substance
?4?2
?3?4
?7
Source
H
N
Me
O
(+)-Anatoxine-a (21)

0.34 2.5 31 Natural
N
Me
O
Me
N-Methylanatoxine (22)

2,600 - > 10,000 Synthetic
N
Me
O
O
Me
Ecgonidine methylic
ester (23)

8,900 - - Synthetic
N
Me
O
Me
(+)-Ferruginina (24)

7,600 - - Natural

Table 4.

Selected Agonists and Antagonist Structurally Related to 25
Ki (nM)
Substance
?4?2
?3?4
?7
Source
N
N
Anabaseine (25)

8 - 280 Natural
N
N
(26)

59 - - Synthetic
N
N
OCH3
OCH3
GTS-21 (27)

20 * - 650 Synthetic

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Advances in Drug Discovery to Assess Cholinergic Neurotransmission Current Drug Discovery Technologies, 2008, Vol. 5, No. 3 7
(Table 4). Contd…..
Ki (nM)
Substance
?4?2
?3?4
?7
Source
N
N
N(CH3)2
(28)

* Antagonist
110 * - 140 Synthetic

Table 5.

Selected Agonists and Antagonist Structurally Related to 4
Ki (nM)
Substance
?4?2
?3?4
?7
Source
NCl
H
N
(-)-Epibatidine (4)

0.058 0.51 21 Natural
H
N
O
N
Me
Epiboxidine (29)

0.46 15 - Synthetic
H
N
N
Cl
(±)-UB-165 (30)

0.27 6.5 2,800 Synthetic

Table 6.

Selected Agonists Structurally Related to Pyridilethers
Ki (nM)
Substance
?4?2
?3?4
?7
Source
N
Me
O
N
A-84543 (31)

0.15 32 2,700 Synthetic
N
H
O
N
Cl
(32)

0.090 - - Synthetic
N
Cl
O
N
H
(33)

0.040 - - Synthetic

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8 Current Drug Discovery Technologies, 2008, Vol. 5, No. 3
Ferreira et al.
Table 7.

Selected Agonists and Antagonist Structurally Related to 1
Ki (nM)
Substance
M2
M3
Source
N
O
Me
Me
Me
Me
O
NH2
Cl
(S) -Bethanechol (10)

1,479 309 Synthetic
N
OH
Diphenidol (35)

190* 95* Synthetic
N
OCH3
O
(36)

25* 114* Synthetic
N
S
O
O
O
Me
(37)

* Antagonist
6,309* 954* Synthetic

pyridilether derivative with a chlorine atom at position 6 on the pyri-
dine ring led to compound 32 with high affinity for the ?4?2 receptor.
Replacing pyrrolidine ring with an azetidine molecule (33) increased
the affinity for the ?4?2 receptor two fold.
AGONISTS AND ANTAGONISTS OF MUSCARINIC RE-
CEPTORS
This section will present some examples of muscarinic receptor
agonists and antagonists, classified according to their class or similar-
ity to natural products. It will also present the affinity constant values
(Ki) of each compound for the corresponding muscarinic receptor
subtypes.
Acetylcholine (1) Analogs
Acetylcholine analogs are an important class of compounds that
act on muscarinic receptors [43, 57]. Several substances of this class
are shown in the Table 7. (S)-bethanechol (10) is a high affinity mus-
carinic agonist but it does not have strong affinity for any specific
receptor subtype. The insertion of bulky groups, such as phenyl, led
to compounds with a better muscarinic receptor antagonist profile, for
instance diphenidol [58] (35).
The transformation of the hydroxyl group in 35 into methyl ether,
as well as the insertion of a carbonyl group close to this functionality
led to 36, which shows high affinity for the M2 receptor subtype. On
the other hand, substituting the hydroxyl group in 35 with a sulphone
produced 37, which had a reduced affinity for the M2 receptor sub-
type [58].
TROPANIC ALKALOIDS ANALOGS
The substances in this class are structurally related to the alkaloid
atropine (8, Table 8), which is found in Atropa belladonna. These
alkaloids act as muscarinic receptor antagonists without a demon-
strated selectivity for any specific receptor subtype.
Benztropine (38) was prepared from 8 by modifications in the es-
ter portion. These structural changes increase its affinity and selectiv-
ity for the M1 receptor subtype [59].
PIPERAZINES AND PIPERIDINES ANALOGS
Piperazine diphenyl sulphoxides belong to a class of muscarinic
antagonist compounds with a high affinity for muscarinic these recep-
tors [60], specifically for the M1 and M2 subtypes, like substance 39
in the Table 9 [61].
Substance 40 differs from 39 only in the substitution of the
piperazinic ring with a piperidinic ring. It has a stronger affinity for
all muscarinic receptor subtypes than 39, but it keeps its antagonist
profile without selectivity for any receptor subtype. The insertion of a
carbonyl group substituting the methyl group in 40 as well as the
substitution of sulphinyl with the sulphonyl group dramatically re-
duced the affinity for muscarinic receptors in 42. It is important to
emphasize that the sulphones 39 and 40 were used as a racemic mix-
ture.

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Advances in Drug Discovery to Assess Cholinergic Neurotransmission Current Drug Discovery Technologies, 2008, Vol. 5, No. 3 9
Table 8.

Selected Antagonist Structurally Related to Tropanic Alkaloids
Ki (nM)
Substance
M1
M2
M3
M4
M5
Source
N
Me
O
O
OH
H
(±)-Atropine (8)

0.50 0.90 1.1 0.6 1.7 Natural
N
Me
O
H
Benztropine (38)

0.231 1.4 1.1 1.1 2.8 Synthetic

Table 9.

Selected Antagonists Structurally Related to Piperazines and Piperidines
Ki (nM)
Substance
M1
M2
M3
M4
M5
Source
OCH3
S
O
N
Me
N
(39)

6.0 1.0 0.97 2.2 - Synthetic
H3CO
S
O
Me
N
(40)

0.53 0.14 0.16 0.07 1.66 Synthetic

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10 Current Drug Discovery Technologies, 2008, Vol. 5, No. 3
Ferreira et al.
(Table 9). Contd…..
Ki (nM)
Substance
M1
M2
M3
M4
M5
Source
H3CO
S
O
O
N
O
(41)

31.09 3.07 13.76 9.05 - Synthetic

Tolterodine Analogs (42)
Tolterodine (42, Table 10) is a synthetic muscarinic antagonist
that is indicated for the treatment of urinary incontinency [62] since it
exhibits affinity for muscarinic receptors.
Cyclic amine 43 had less affinity for receptors M2 and M3 than
did tolterodine. Substitution of a hydroxyl group in 43 with benzylic
ether produced a compound, with very low affinity for muscarinic
receptors when compared to substance 43 [63].
Oxibutinine Analogs (45)
Oxibutinine (45, Table 11) is an aminoalcohol that is structurally
related to procyclidine (12). Clinical assays have demonstrated its
effectiveness in treating urinary incontinency [64-67], probably due
to its high affinity and selectivity for the M3 muscarinic receptor sub-
type.
The insertion of a cyclic amine to replace the aliphatic amine pre-
sent in 45 greatly decrease the muscarinic activity of the compound,
as observed in 46. Meanwhile, substituting the cyclohexyl ring with a
phenyl group, as observed in 47, slightly increased the affinity for
muscarinic receptors, when compared to 46; however, this affinity is
considerably less than that of oxibutinine (45) [63].
QUINUCLIDINES ANALOGS
The synthesis of substances having a quinuclidinic ring and terci-
ary alcohol portion is an excellent method for obtaining compounds
Table 10.

Selected Antagonists and Antagonist Structurally Related to 42
Ki (nM)
Substance
M2
M3
Source
OH
Me
NMe
Me
MeMe
Tolterodine (42)

6.91 7.07 Synthetic
OH
Me
N
H
H
Me
(43)

42 25 Synthetic
O
Me
N
Me
Me
(44)

> 10,000 > 10,000 Synthetic

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Advances in Drug Discovery to Assess Cholinergic Neurotransmission Current Drug Discovery Technologies, 2008, Vol. 5, No. 3 11
with high affinity for muscarinic receptors. Some substances of this
class are presented in Table 12.
Compound 48 shows very high affinity for the M2 and M3 mus-
carinic receptor subtypes. Its affinity was increased by the substitu-
tion of a phenyl group by a thienyl group, but it has less selectivity
than 49. On the other hand, the insertion of a methyl group at position
4 of the phenyl groups (48) reduced the affinity for the M2 and M3
muscarinic receptor subtypes (50) [68].
Muscarine (2) Analogs
Muscarine (2, Table 13) is the main alkaloid found in the poison
toadstool Amanita muscaria. Its strong cholinomimetic activity [16,
69, 70] confers great interest on this substance and its analogs. Mus-
carine (2) shows a moderate affinity, without any selectivity, for the
M2 e M3 receptor subtypes.
Angeli and coworkers [71] synthesised several analogs of 2 that
act on muscarinic receptors. Substitution of the hydroxyl group in 2
with a fluorine atom increases affinity for the M3 receptor subtype,
creating 51. However, inserting a second fluorine atom decreases the
affinity (52).
PERSPECTIVES
As indicated in the previous sections, the two major neurodegen-
erative diseases, Alzheimer’s disease (AD) and Parkinson’s disease
(PD), are characterized by low levels of the neurotransmitters acetyl-
choline (ACh) and dopamine (DA) in brain. The available clinical
treatments for these two conditions are palliative and rely, in these
most cases, on improving stimulation at the receptors by either in-
creasing the levels of the endogenous neurotransmitter or by the use
of substances that have a similar agonist response.
In this regard, and as well as supplying useful drugs in their own
right, natural products also provide templates for the development of
other compounds. For example, scientific literature is full of natural
products from plant and marine organisms that are able to act on di-
verse pathophysiological conditions [72].
We feel that investigating natural products to identify new candi-
date molecules that might act on cholinergic neurotransmission may
be of great importance in designing new therapeutic approaches.
In particular, cholinergic neurotransmission has been a target for
studies that search for new prototypes to act at this critical physiol-
ogic level [47].
We have also been dedicating efforts into researching on natural
products that target cholinergic neurotransmission. We are investigat-
ing cyanobacteria [73] and a plant called Eugenia punicifolia [74].
Cyanobacteria are photosynthetic prokaryotes that are widely
employed in food by humans. They have been recognised as a useful
source of vitamins and proteins and are also reported to be a source of
fine chemicals, renewable fuel and bioactive compounds [75]. Cya-
nobacteria possess a wide range of colored compounds, such as caro-
tenoids, chlorophyll, and phycobiliproteins. The main phycobilipro-
teins are C-phycocyanin (C-PC), allo-phycocyanin, and phyco-
erythrin, which are blue and red coloured proteins involved in energy
absortion in photosynthesis [76]. C-phycocyanin has been shown to
have hepatoprotective [77], anti-inflammatory [78], and antioxidant
properties [79]. It was also demonstrated that C-PC has potential as a
novel antiplatelet agent to treat of arterial thromboembolism [80].
Cyclic peptides that can inhibit cell growth have been isolated from a
marine cyanobacterium Anabaena torulosa. The structures of the two
major components were identified as laxaphycin A and B. The anti-
proliferative activity of these laxaphycins was investigated on a panel
of solid and lymphoblastic cancer cells; laxaphycin A alone did not
affect proliferation but laxaphycin B did inhibit the proliferation of
sensitive and resistant human cancer cell lines and the presence of
laxaphycin A strongly enhanced this effect [81]. A new quaternary
beta-carboline alkaloid, nostocarboline, was isolated from the fresh-
water cyanobacterium Nostoc 78-12A; 2D-NMR determination of its
structure was confirmed by its successful synthesis. Nostocarboline
was found to be a potent inhibitor for butyrylcholinesterase, one of
the enzymes responsible for the biochemical degradation of acetyl-
choline in cholinergic nervous terminations, and that has an IC50 of
13.2 ?M. This inhibitory concentration is comparable to that of gal-
anthamine, a drug approved for the treatment of Alzheimer’s disease.
Table 11.

Selected Agonists and Antagonist Structurally Related to 45
Ki (nM)
Substance
M2
M3
Source
OH
O
NMe
Me
O
(±)-Oxibutinine (45)

6.97 0.95 Synthetic
OH
H
N
N
O
(46)

3,582 2,605 Synthetic
OH
H
N
N
O
(47)

639 291 Synthetic

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12 Current Drug Discovery Technologies, 2008, Vol. 5, No. 3
Ferreira et al.
Table 12.

Selected Agonists Structurally Related to Quinuclidines
Ki (nM)
Substance
M2
M3
Source
N
MeO
HO
(48)

0.79 0.13 Synthetic
S
S
N
MeO
HO
(49)

0.20 0.05 Synthetic
N
MeO
HO
Me
Me
(50)

501 100 Synthetic

Table 13.

Selected Agonists Structurally Related to 2
Ki (nM)
Substance
M2
M3
Source
O
Me
HO
N
Me
Me
Me
Cl
(±)-Muscarine (2)

204 79 Natural
O
(51)
Me
F
N
Me
Me
Me
Cl

186 43 Synthetic
O
Me
F
N
Me
Me
Me
F
Cl
(52)

275 1,584 Synthetic

Thus, nostocarboline can be considered a possibility in the search to
novel neurochemicals to treat neurodegeneration [82]. Furthermore,
Cox and co-workers [83] have shown that ?-N-methylamino-L-
alanine (BMAA), a nonprotein amino acid, was produced by cyano-
bacterial root symbionts of the genus Nostoc. Since BMAA was re-
ported to be present in the brain tissues of some Canadian Alz-
heimer’s patients [84], there is a strong possibility of connections
between this cyanobacteria and AD. These findings have brought to
our group important research perspectives, since we have demon-
strated that culture conditions can modify antioxidant metabolite
biosynthesis in the cyanobacterium Anabaena PCC7119 [73], and we
are presently testing these metabolites in our laboratory. We really

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Advances in Drug Discovery to Assess Cholinergic Neurotransmission Current Drug Discovery Technologies, 2008, Vol. 5, No. 3 13
feel that cyanobacteria provide useful tools for studying cholinergic
neurotransmission.
On the other hand, the genus Eugenia has already produced some
important results in some experimental models, indicating the possi-
ble use of some species to treat in chronic diseases, such as hyperten-
sion and diabetes mellitus [85-88]. Some of the major chemicals ac-
tive components isolated from the genus include flavonoids, essential
oils, phenolic coumponds, steroids and triterpenoids, among others
[88].
We recently demonstrated that an aqueous extract of the plant E.
punicifolia, popularly known in Brasil as “pedra-ume caá” or “very
wet stone”, was able to change the response pattern of some nicotinic
antagonists in the rat diaphragm, as well as to potentiate cholinergic
nicotinic neurotransmission. Thus, our results indicated that products
from this plant could serve as a new pharmacological tool for assess-
ing mechanisms involved in cholinergic nicotinic neurotransmission.
We believe that the description of new, natural pharmacological
tools for studying cholinergic neurotransmission, like E. punicifolia
or Anabaena PCC7119, can help to understand some diseases in
which cholinergic neurotransmission is implicated and also in the
design of therapeutic strategies for diseases with impaired cholinergic
neurotransmission.
CONCLUSION
The data presented in this review reflect the great importance of
cholinergic neurotransmission in physiology and several diseases.
Identification of substances that can modulate this system should
advance our understanding of neurophisiopathology.
Up to now no compounds have been identified that bind selec-
tively or specifically to muscarine receptors. Identifying substances
with these features as well as new agonists and antagonists for nico-
tinic receptors, will allow us to create drugs that are more potent and
selective than those now available as well as to obtain more informa-
tion about the nature of nicotinic and muscarinic receptors. These
discoveries could also be important for the treatment of diseases that
affect a large number of people, like Parkinson’s disease, Alzheimer’s
disease and myasthenia gravis.
As presented in this work, the use of several natural products
constitutes an excellent alternative to the synthesis of compounds
with activity on cholinergic neurotransmission. Its main attractions
are the low cost and the possibility of characterizing chiral substrates
with a known absolute configuration. A good synthetic design will
take advantage of the chiral centers present in natural compounds in
order to obtain enantiomerically pure products which are a character-
istic of bio-actives substances.
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