Current Drug Discovery Technologies, 2008, 5, 000-000
1
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 1
Departamento de Química Orgânica, Instituto de Química, Universidade Federal Fluminense, Outeiro de S. João Batista s/n Centro 24020-150, Niterói, RJ, Brazil 2
Departamento de Bromatologia, Faculdade de Farmácia, Universidade Federal Fluminense, Rua Mário Viana 523, Santa Rosa 24241-000, Niterói, RJ, Brazil 3 Departamento 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: 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.
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 curiosity. 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 functions and may determine many disorders and diseases, still need to be better understood. Continued efforts to study the mechanisms controlling 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, muscular 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 neurotransmission. NEUROTRANSMISSION Neurotransmission is the process whereby nervous system cells communicate among themselves and with others cells of the organism through nerve impulses. This process produces responses in smooth, cardiac and skeletal muscles and glands [4]. This fundamental physiological event is mediated by some specific chemical compounds called neurotransmitters. Many of them are involved in diverse body functions: they include noradrenaline, dopamine and *Address correspondence to this author at the Departamento de Química Orgânica, Instituto de Química, Universidade Federal Fluminense, Outeiro de S. João Batista s/n Centro 24020-150, Niterói, RJ, Brazil; E-mail:
[email protected]
1570-1638/08 $55.00+.00
histamine [4]. Acetylcholine (ACh, 1, Fig. (1)), the first neurotransmitter 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]. O
Me Me N
Me
O
Me
Cl
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 precursors. 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 essential for proper muscle function [4]. Once released, the neurotransmitter (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 cholinergic receptors: a) muscarinic receptors (M), which belong to the metabotropic receptor family; and b) nicotinic receptors (N), which belong to the ionotropic receptor family [16]. This nomenclature originated in the observation that acetylcholine mimicked the pharmacological 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 [1821] (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 © 2008 Bentham Science Publishers Ltd.
2 Current Drug Discovery Technologies, 2008, Vol. 5, No. 3
Ferreira et al.
Fig. (2). Schematic illustration of cholinergic neurotransmission.
range of possible subunit combinations. Until now, ten types of subunits and four nicotinic receptors have been described as composing the receptor sites [18, 22]. Depending on their subunit combination, nicotinic receptors show different activation and inactivation kinetics and variable physiopharmacological properties [14]. OH Cl Me
Me N
N
Me O
Me
Muscarine (2)
Me
N
Nicotine (3)
Fig. (3). Alkaloids muscarine (2) and nicotine (3).
This diverse composition suggests the existence of a large number 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 functions [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 functions, such as analgesia, neurotransmitter release and neuroprotection [27].
Fig. (4). Schematic illustration of nicotinic receptor.
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 instance, 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, pancuronium (6) and rocuronium (7), inhibit ACh binding to receptors [30]. These substances, and other analogues, are widely employed as neuromuscular in anesthesia. Neuromuscular blocking drugs relax the skeletal muscles and induce paralysis [4]. Recently, experiments using bovine adrenal cells as experimental model evidenced the direct action of atropine (8) on nicotinic receptors at nanomolar level concentrations: 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
Advances in Drug Discovery to Assess Cholinergic Neurotransmission
Current Drug Discovery Technologies, 2008, Vol. 5, No. 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. Cl
N
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 muscarinic 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.
Me
H N
N
N Me
(-)-Epibatidine (4)
I
DMPP (5)
Fig. (5). Examples of nicotinic receptors agonists.
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, glandular secretion, pupil dilatation and food intake are mainly mediated by M3 receptors, whilst the effects on cardiac rate are mainly mediated by M2 subtypes [41, 42].
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 understanding of these receptors [4]. O
O Me Me
O
Me Me
Me
Me
H
Me N
Me H
N
H
N
Br
Br
H
O H O
H
HO H
Me Pancuronium (6)
NMe
Rocuronium (7)
H
O OH O Atropine (8)
Fig. (6). Examples of nicotinic receptor antagonists.
Fig. (7). Schematic illustration of muscarinic receptor.
O
O
N H
3
Br
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]. Me Me
Me O
Me Me
N
N Me
N
O
Nicotine (3) Analogs O
NH2
Cl O
Pilocarpine (9)
(S) -Bethanechol (10)
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]. O Me
N
OH OH
ClH N
N (±)Tropicamide (11)
(R)-Procyclidine (12)
Fig. (9). Examples of muscarinic receptors antagonists.
As previously mentioned, muscarinic receptors (1) play a fundamental 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 NEUROTRANSMISSION
ON
choline in that its alcohol function is transformed into carbamate, thus increasing its affinity for nicotinic receptor subtypes, particularly the 42 receptors. The transformation of the carbamylcholine carbamate into N-methylcarbamate (e.g., 15) strong increases the affinity of this substance for 42 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 42 receptors.
Many synthetic compounds that are structurally related to nicotine (3) have been described in the literature. Some of them are described in Table 2. Nicotine (3) is a natural alkaloid found in many plants. The compound shows modest affinity for the 42 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 Nmethyl 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 quinuclidinic ring, but this does not change the affinity for the 42 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 42 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 receptors.
CHOLINERGIC
Given the great variety of nicotinic and muscarinic receptor subtypes, the synthesis of substances that selectively bind to these receptors 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].
Anabaseine (25) Analogs Anabaseine (25, Table 4) is a natural toxin produced by marine 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 42 receptor, despite being potent receptor 7agonists.
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 42, 34 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 42 and 7 receptors subtypes are found in the central nervous system while the 34 subtype is found in ganglious 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 explained by its fast desensitisation. Carbamylcholine (14) differs from
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 affinity for 42 e 34 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 receptors. For instance, pyridilether 31 (Table 6) shows high affinity for the 42 nicotinic receptors. Demethylating the pyrrolidine ring on the
Advances in Drug Discovery to Assess Cholinergic Neurotransmission Table 1.
Current Drug Discovery Technologies, 2008, Vol. 5, No. 3
Selected Agonists Structurally Related Acetylcholine (1) and their Action on the Nicotinic Receptors [47] Ki (nM)
Substance
Source
42
34
7
7,000
-
180,000
Natural
61
1,300
22,000
Synthetic
2.0
460
11,000
Synthetic
18
220
7,600
Synthetic
0.00068
-
-
Synthetic
Me Me
OH N Me
Cl Choline (13)
Me Me
O
NH2
N Cl
Me
O
Carbamylcholine (14)
Me Me
H N
O N
Me
Me
Cl
O
N-Methylcarbamylcholine (15)
Me N
N Me I
DMPP (5) OCH3 HN N
Cl N
(16)
Table 2.
Selected Agonists and Antagonists Structurally Related to 3 Ki (nM) Substance
Source
N
42
34
7
1,0
73
1,600
Natural
0.63
-
-
Synthetic
24
-
36,000
Synthetic
22 *
-
-
Synthetic
1.0 *
-
-
Synthetic
Me
N Nicotine (3)
N N Me 6-Chloronicotine (17)
Me
Cl
N N H
Metanicotine (18)
N N Me
(19)
N
N
* Antagonist
5
6 Current Drug Discovery Technologies, 2008, Vol. 5, No. 3 Table 3.
Ferreira et al.
Selected Agonists Structurally Related to Tropanic Alkaloids Ki (nM)
Substance
Source
42
34
7
0.34
2.5
31
Natural
2,600
-
> 10,000
Synthetic
8,900
-
-
Synthetic
7,600
-
-
Natural
O
H N
Me
(+)-Anatoxine-a (21)
Me
O
N Me
N-Methylanatoxine (22) Me O
N
Me O
Ecgonidine methylic ester (23)
Me O
N
Me
(+)-Ferruginina (24)
Table 4.
Selected Agonists and Antagonist Structurally Related to 25 Ki (nM)
Substance
N
Source
42
34
7
8
-
280
Natural
59
-
-
Synthetic
20 *
-
650
Synthetic
N Anabaseine (25)
N N
(26) OCH3
OCH3
N N GTS-21 (27)
Advances in Drug Discovery to Assess Cholinergic Neurotransmission
Current Drug Discovery Technologies, 2008, Vol. 5, No. 3
7
(Table 4). Contd….. Ki (nM)
Substance
42
34
7
110 *
-
140
Source
N(CH3)2
Synthetic
N (28)
N
* Antagonist
Table 5.
Selected Agonists and Antagonist Structurally Related to 4 Ki (nM) Substance
Cl
Source 42
34
7
0.058
0.51
21
Natural
0.46
15
-
Synthetic
0.27
6.5
2,800
Synthetic
H N
N
(-)-Epibatidine (4)
H N N
O
Me Epiboxidine (29) Cl H N
N
(±)-UB-165 (30)
Table 6.
Selected Agonists Structurally Related to Pyridilethers Ki (nM)
Substance
Source
42
34
7
0.15
32
2,700
Synthetic
0.090
-
-
Synthetic
0.040
-
-
Synthetic
O N N
Me A-84543 (31)
O N H
N (32)
Cl
O N N H
Cl
(33)
8 Current Drug Discovery Technologies, 2008, Vol. 5, No. 3 Table 7.
Ferreira et al.
Selected Agonists and Antagonist Structurally Related to 1 Ki (nM) Substance
Source M2
M3
1,479
309
Synthetic
190*
95*
Synthetic
25*
114*
Synthetic
6,309*
954*
Synthetic
Me Me Me
N Me
O
NH2
Cl O (S) -Bethanechol (10)
OH N
Diphenidol (35)
OCH3 N O (36) Me O S O N O (37) * Antagonist
pyridilether derivative with a chlorine atom at position 6 on the pyridine ring led to compound 32 with high affinity for the 42 receptor. Replacing pyrrolidine ring with an azetidine molecule (33) increased the affinity for the 42 receptor two fold. AGONISTS AND ANTAGONISTS OF MUSCARINIC RECEPTORS This section will present some examples of muscarinic receptor agonists and antagonists, classified according to their class or similarity 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 muscarinic 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 subtype [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 demonstrated selectivity for any specific receptor subtype. Benztropine (38) was prepared from 8 by modifications in the ester portion. These structural changes increase its affinity and selectivity 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 receptors [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 reduced the affinity for muscarinic receptors in 42. It is important to emphasize that the sulphones 39 and 40 were used as a racemic mixture.
Advances in Drug Discovery to Assess Cholinergic Neurotransmission Table 8.
Current Drug Discovery Technologies, 2008, Vol. 5, No. 3
Selected Antagonist Structurally Related to Tropanic Alkaloids Ki (nM) Substance
Source M1
M2
M3
M4
M5
0.50
0.90
1.1
0.6
1.7
Natural
0.231
1.4
1.1
1.1
2.8
Synthetic
Me N
H
O OH O (±)-Atropine (8)
Me N
H O
Benztropine (38)
Table 9.
Selected Antagonists Structurally Related to Piperazines and Piperidines Ki (nM) Substance
Source M1
M2
M3
M4
M5
6.0
1.0
0.97
2.2
-
Synthetic
0.53
0.14
0.16
0.07
1.66
Synthetic
Me N O
N S
(39) OCH3 Me N
O
H3CO
S
(40)
9
10 Current Drug Discovery Technologies, 2008, Vol. 5, No. 3
Ferreira et al.
(Table 9). Contd….. Ki (nM) Substance
Source M1
M2
M3
M4
M5
31.09
3.07
13.76
9.05
-
O N
O
S
Synthetic
O
(41) H3CO
Tolterodine Analogs (42)
effectiveness in treating urinary incontinency [64-67], probably due to its high affinity and selectivity for the M3 muscarinic receptor subtype.
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.
The insertion of a cyclic amine to replace the aliphatic amine present 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].
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)
QUINUCLIDINES ANALOGS
Oxibutinine (45, Table 11) is an aminoalcohol that is structurally related to procyclidine (12). Clinical assays have demonstrated its Table 10.
The synthesis of substances having a quinuclidinic ring and terciary alcohol portion is an excellent method for obtaining compounds
Selected Antagonists and Antagonist Structurally Related to 42 Ki (nM)
Substance OH
Me
Source
M2
M3
6.91
7.07
Synthetic
42
25
Synthetic
> 10,000
> 10,000
Synthetic
Me N
Me
Me Me
Tolterodine (42) H OH N Me
H Me (43)
Me O N Me
Me
(44)
Advances in Drug Discovery to Assess Cholinergic Neurotransmission Table 11.
Current Drug Discovery Technologies, 2008, Vol. 5, No. 3
11
Selected Agonists and Antagonist Structurally Related to 45 Ki (nM) Substance
Source
OH
N
M2
M3
6.97
0.95
Synthetic
3,582
2,605
Synthetic
639
291
Synthetic
Me
O Me O (±)-Oxibutinine (45)
OH
N
H N O
(46)
OH
N
H N O (47)
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 muscarinic receptor subtypes. Its affinity was increased by the substitution 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. Muscarine (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 neurodegenerative diseases, Alzheimer’s disease (AD) and Parkinson’s disease (PD), are characterized by low levels of the neurotransmitters acetylcholine (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 increasing 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 diverse pathophysiological conditions [72].
We feel that investigating natural products to identify new candidate 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 physiologic level [47]. We have also been dedicating efforts into researching on natural products that target cholinergic neurotransmission. We are investigating 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]. Cyanobacteria possess a wide range of colored compounds, such as carotenoids, chlorophyll, and phycobiliproteins. The main phycobiliproteins are C-phycocyanin (C-PC), allo-phycocyanin, and phycoerythrin, 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 antiproliferative 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 freshwater 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 acetylcholine in cholinergic nervous terminations, and that has an IC50 of 13.2 M. This inhibitory concentration is comparable to that of galanthamine, a drug approved for the treatment of Alzheimer’s disease.
12 Current Drug Discovery Technologies, 2008, Vol. 5, No. 3 Table 12.
Ferreira et al.
Selected Agonists Structurally Related to Quinuclidines Ki (nM) Substance
Source M2
M3
0.79
0.13
Synthetic
0.20
0.05
Synthetic
501
100
Synthetic
MeO N HO
(48) MeO N HO S S (49)
MeO N HO
Me Me
(50)
Table 13.
Selected Agonists Structurally Related to 2 Ki (nM) Substance
Source M2
M3
204
79
Natural
186
43
Synthetic
275
1,584
Synthetic
HO Me
Me N
Me
Me
O
Cl (±)-Muscarine (2) F Me
Me N
Me
O (51)
Me Cl
F F
Me
Me N
Me
Me
O (52)
Cl
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-Lalanine (BMAA), a nonprotein amino acid, was produced by cyanobacterial root symbionts of the genus Nostoc. Since BMAA was reported 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 demonstrated 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
Advances in Drug Discovery to Assess Cholinergic Neurotransmission
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 possible use of some species to treat in chronic diseases, such as hypertension and diabetes mellitus [85-88]. Some of the major chemicals active 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 assessing 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
Current Drug Discovery Technologies, 2008, Vol. 5, No. 3
[10]
[11] [12]
[13] [14] [15] [16] [17] [18]
[19] [20]
[21]
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.
[22]
Up to now no compounds have been identified that bind selectively or specifically to muscarine receptors. Identifying substances with these features as well as new agonists and antagonists for nicotinic receptors, will allow us to create drugs that are more potent and selective than those now available as well as to obtain more information 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.
[24]
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 characteristic of bio-actives substances.
[23]
[25]
[26]
[27]
[28]
[29]
[30]
REFERENCES [1] [2]
[3] [4] [5] [6]
[7]
[8]
[9]
Bonuccelli U., Del Dotto P.: New pharmacologic horizons in the treatment of Parkinson disease. Neurology 67(7), S30-S38, (2006). Kepe V., Huang S.C., Small G.W., Satyamurthy N., Barrio J.R.: Visualizing pathology deposits in the living brain of patients with Alzheimer’s disease. Methods Enzimol. 412, 144-160, (2006). Benatar M.: A systematic review of diagnostic studies in myasthenia gravis. Neuromuscul. Disord. 16(7), 459-467, (2006). Brunton L.L.; Lazo J.S.; Parker K.L. Goodman & Gilman’s The Pharmacological Basis of Therapeutics, 11th Ed., McGraw-Hill, New York, (2006). Starke K., Gothert M., Kilbinger H.: Modulation of neurotransmitter release by presynaptic autoreceptors. Physiol. Rev. 69(3), 864-989, (1989). Woody C.D., Gruen E.: Acetylcholine reduces net outward currents measured in vivo with single electrode voltage clamp techniques in neurons of the motor cortex of cats. Brain. Res. 424(1), 193-198, (1987). Bucci D.J., Holland P.C., Gallagher M.: Removal of cholinergic input to rat posterior parietal cortex disrupts incremental processing of conditioned stimuli. J. Neurosci. 18(19), 8038-8046, (1998). Voytko M.L., Olton D.S., Richardson R.T., Gorman L.K., Tobin J.R., Price D.L.: Basal forebrain lesions in monkeys disrupt attention but not learning and memory. J. Neurosci. 14(1), 167-186, (1994). Fine A., Hoyle C., Maclean C.J., Levatte T.L., Baker H.F., Ridley R.M.: Learning impairments following injection of a selective cholinergic immuno-
[31]
[32]
[33]
[34]
[35]
[36]
[37]
13
toxin, ME20.4 IgG-saporin, into the basal nucleus of Meynert in monkeys. Neuroscience 81(2), 331-343, (1997). Hasselmo M.E., Anderson B.P., Bower J.M.: Cholinergic modulation of cortical associative memory function. J. Neurophysiol. 67(5), 1230-1246, (1992). Sarter M., Bruno J.P.: Cognitive functions of cortical acetylcholine: Toward a unifying hypothesis. Brain. Res. Rev. 23(1-2), 28-46, (1997). Newman M.B., Nazian S.J., Sanberg P.R., Diamond D.M., Shytle R.D.: Corticosterone-attenuating and anxiolytic properties of mecamylamine in the rat. Prog. Neuropsycopharmacol. Biol. Psychiatr. 25(3), 609-620, (2001). Guyton A.C., Hall J.E. Tratado de Fisiologia Médica, 10ª Ed., Guanabara: Rio de Janeiro, (2002). Racké K., Matthiessen S.: The airway cholinergic system: physiology and pharmacology. Pulm. Pharmacol. Ther. 17(4), 181-198, (2004). Rang H.P.: The receptor concept: pharmacology’s big idea. Br. J. Pharmacol. 147, S9-S16, (2006). Dale H.H.: The action of certain esters and ethers of choline, and their relation to muscarine. J.Pharmacol.Exp Ther. 6, 147-190, (1914). Lucas-Meunier E., Fossier P., Amar M.: Cholinergic modulation of the cortical neuronal network. Eur. J. Physiol. 446(1), 17-29, (2003). Conti-Tronconi B.M., McLane K.E., Raftery M.A., Grando S.A., Protti M.P.: The nicotinic acetylcholine-receptor - structure and autoimmune pathology. Crit. Rev. Biochem. Mol. Biol. 29(2), 69-123, (1994). Galzi J.L., Changeux J.P.: Neuronal nicotinic receptors – molecularorganization and regulations. Neuropharmacology 34(6), 563-582, (1995). Lukas R.J., Changeux J.P., Le Novere N., Albuquerque E.X., Balfour D.J., Berg D. K., Bertrand D., Chiappinelli V.A., et al.: International Union of Pharmacology. XX. Current status of the nomenclature for nicotinic acetylcholine receptors and their subunits. Pharmacol. Rev. 51(2), 397-401, (1999). Lindstrom J.M.: Acetylcholine receptors and myasthenia. Muscle Nerve 23(4), 453-477, (2000). Sgard F., Charpantier E., Bertrand S., Walker N., Caput D., Graham D., Bertrand D., Besnard F.: A novel human nicotinic receptor subunit, alpha 10, that confers functionality to the alpha 9-subunit. Mol. Pharmacol. 61(1), 150159, (2002). Steinlein O.: New functions for nicotinic acetylcholine receptors? Behav. Brain Res. 95(1), 31-35, (1998). Clementi F., Fornassari D., Gotti C.: Neuronal nicotinic receptors, important new players in brain function. Eur. J. Pharmacol. 393(1-3), 3-10, (2000). Cordero-Erausquim M., Marubio L.M., Klink R., Changeux J.P.: Nicotinic receptor function: new perspectives from knockout mice. Trends Pharmacol. Sci. 21(6), 211-217, (2000). Perry E.K., Morris C.M., Court J.A., Cheng A., Fairbain A.F., McKeith I.G., Irving D., Brown A., et al.: Alteration in nicotine binding-sites in parkinsonsdisease, lewy body dementia and alzheimers-disease – possible index of early neuropathology. Neuroscience 64(2), 385-395, (1995). Romanelli M.N., Manetti D., Scapecchi S., Borea P.A., Dei S., Bartolini A., Ghelardini C., Gualtieri F., et al.: Structure-affinity relationships of a unique nicotinic ligand: N-1-dimethyl-N-4-phenylpiperazinium iodide (DMPP). J. Med.Chem. 44(23), 3946-3955, (2001). González-Rubio J.M., Rojo J., Tapia L., Maneu V., Mulet J., Valor L.M., Criado M., Sala F., et al.: Activation and blockade by choline of bovine alpha 7 and alpha 3 beta 4 nicotinic receptors expressed in oocytes. Eur. J. Pharmacol. 535(1-3), 53-60, (2006). Carrol F.I., Ma W., Yokota Y., Lee J.R., Brieaddy L.E., Navarro H.A., Damaj M. I., Martin B.R.: Synthesis, nicotinic acetylcholine receptor binding, and antinociceptive properties of 3’-substituted deschloroepibatidine analogues. Novel nicotinic antagonists. J. Med.Chem. 48(4), 1221-1228, (2005). Rang H.P., Dale M.M., Ritter J.M., Moore P.K.: Pharmacology; 5th Ed., Churchill Livingstone: Edinburgh, (2003). González-Rubio J.M., de Diego A.M.G., Egea J., Olivares R., Rojo J., Gandía A. G., Hernandéz-Guijo J.M.: Blockade of nicotinic receptors of bovine adrenal chromaffin cells by nanomolar concentrations of atropine. Eur. J. Pharmacol. 535(1-3), 13-24, (2006). Connor E.A., Levy S.M., Parsons R.L.: Kinetic-analysis of atropine induced alterations in bullfrog ganglionic fast synaptic currents. J. Physiol. 337(4), 137-158, (1983). Elgoyhen A.B., Johnson D.S., Boulter J., Vetter D.E., Heinemann S.F.: Alpha-9 – an acetylcholine-receptor with novel pharmacological properties expressed in rat cochlear hair-cells. Cell 79(4), 705-715, (1994). Parker J.C., Sarkar D., Quick M.W., Lester R.A.: Interactions of atropine with heterologously expressed and native alpha 3 subunit-containing nicotinic acetylcholine receptors. Brit. J. Pharmacol. 138(5), 801-810, (2003). Curtis C.A.M., Wheatley M., Bansal S., Birdsall N.J., Eveleigh P., Pedder E.K., Poyner D., Hulme E.C.: Propylbenzilylcholine mustard labels an acidic residue in transmembrane helix-3 of the muscarinic receptor. J. Biol. Chem. 264(1), 489-495, (1989). Wess J., Blin N., Mutschler E., Bluml K.: Muscarinic acetylcholinereceptors – structural basis of ligand-binding and g-protein coupling. Life Sci. 56(11-12), 915-922, (1995). Bonner T.I., Buckley N.J., Young A.C., Brann M.R.: Identification of a family of muscarinic acetylcholine-receptor genes. Science 237(4814), 527532, (1987).
14 Current Drug Discovery Technologies, 2008, Vol. 5, No. 3 [38]
[39]
[40] [41]
[42]
[43] [44]
[45]
[46]
[47] [48]
[49]
[50] [51] [52] [53] [54]
[55]
[56] [57]
[58]
[59]
[60]
[61]
[62]
[63]
Kubo T., Fukuda K., Mikami A., Maeda A., Takashi H., Mishina M., Haga T., Haga K., et al.: Cloning, sequencing and expression of complementaryDNA encoding the muscarinic acetylcholine-receptor. Nature 323(6087), 411-416, (1986). Kubo T., Maeda A., Sugimoto K., Akiba I., Mikami A., Takahashi H., Haga T., Haga K., et al.: Primary structure of porcine cardiac muscarinic acetylcholine-receptor deduced from the cDNA sequence. FEBS Lett. 209(2), 367372, (1986). Bonner T.I.: The molecular-basis of muscarinic receptor diversity. Trends. Neurosci. 12(4), 148-151, (1989). Caulfield M.P., Birdsall N.J.: International Union of Pharmacology. XVII. Classification of muscarinic acetylcholine receptors. Pharmacol. Rev. 50(2), 279-290, (1998). Bymaster F.P., McKinzie D.L., Felder C.C., Wess J.: Use of M-1-M-5 muscarinic receptor knockout mice as novel tools to delineate the physiological roles of the muscarinic cholinergic system. Neurochem. Res. 28(3-4), 437442, (2003). Broadley K.J., Kelly D.R.: Muscarinic receptor agonists and antagonists. Molecules 6(3), 142-193, (2001). Buchwald P., Bodor N.: Soft quaternary anticholinergics: Comprehensive quantitative structure-activity relationship (QSAR) with a linearized biexponential (LinBiExp) mode. J. Med. Chem. 49(3), 883-891, (2006). Daly J.W., Gupta T.H., Padgett W.L., Pei X.: 6 beta-acyloxy(nor)tropanes: Affinities for antagonist/agonist binding sites on transfected and native muscarinic receptors. J. Med. Chem. 43(13), 2514-2522, (2000). Viegas Jr.,C., Bolzani V.S. Furlan M., Fraga C.A.M., Barreiro E.J.: Produtos naturais como candidatos a fármacos úteis no tratamento do mal de Alzheimer, Quim. Nova 27(4), 655-660, (2004). Daly J.W.: Nicotinic agonists, antagonists, and modulators from natural sources. Cell. Mol. Neurobiol. 25(3-4), 513-552, (2005). Hjelmgaard T., Sotofte I., Tanner D.: Total synthesis of pinnamine and anatoxin- A via a common intermediate. A caveat on the anatoxin-A endgame. J. Org. Chem. 70(14), 5688-5697, (2005). Devlin J.P., Edwards O.E., Gorham P.E., Hunter N.R., Pike R.K., Stavric B.: Anatoxin-A, a toxic alkaloid from anabaena-flos-aquae NRC-44H. Can. J. Chem. 55(8), 1367-1371, (1977). Bick I.R.C., Gillard J.W., Leow H.: Alkaloids of darlingia-darlingiana. Aust. J. Chem. 32(11), 2523-2536, (1979). Bick I.R.C., Gillard J.W., Leow H.: Alkaloids of darlingia-ferruginea. Aust. J. Chem. 32(11), 2537-2543, (1979). Kem W.R., Scott K.N., Duncan J.H.: Hoplonemertine worms – a new Source of pyridine neurotoxins. Experientia 32(6), 684-686, (1976). Wheeler J.W., Olubajo O., Storm C.B., Duffield R.M.: Anabaseine: Venom alkaloid of aphaenogaster ants. Science 211(4486), 1051-1052, (1981). Kem W.R., Mahnir V.M., Papke R.L., Lingle C.J.: Anabaseine is a potent agonist on muscle and neuronal alpha-bungarotoxin-sensitive nicotinic receptors. J. Pharmacol. Exp. Ther. 283(3), 979-992, (1997). Spande T.F., Garrafo H.M., Edwards M.W., Yeh H.J.C., Pannell L., Daly J.W.: Epibatidine – a novel (chloropyridyl)azabicycloheptane with potent analgesic activity from an hinese an poison frog. J. Am. Chem. Soc. 114(9), 3475-3478, (1992). Dukat M., Glennon R.A.: Epibatidine: Impact on nicotinic receptor research. Cell. Mol. Neurobiol. 23(3), 365-378, (2003). De Amici M., Conti P., Fasoli E., Barocelli E., Ballabeni V., Bertoni S., Impicciatore M., Roth B.L., et al.: Synthesis and in vitro pharmacology of novel heterocyclic muscarinic ligands. Il Farmaco 58(9), 739-748, (2003). Varoli L., Angeli P., Burnelli S., Marucci G., Recanatini M.: Synthesis and antagonistic activity at muscarinic receptor subtypes of some 2-carbonyl derivatives of diphenidol. Bioorg. Med. Chem. 7(9), 1837-1844, (1999). Bolden C., Cusack B., Richelson E.: Antagonism by antimuscarinic and neuroleptic compounds at the 5 cloned human muscarinic cholinergic receptors expressed in hinese-hamster ovary cells. J. Pharmacol. Exp. Ther. 260(2), 576-580, (1992). Kozlowski J.A., Lowe D.B., Guzik H.S., Zhou H., Ruperto V.B., Duffy R.A., McQuade R., Crosby G.Jr., et al.: Diphenyl sulfoxides as selective antagonists of the muscarinic M-2 receptor. Bioorg. Med. Chem. Lett. 10(20), 2255-2257, (2000). Billard W., Binch III H., Bratzler K., Chen L., Crosby Jr. G., Duffy R.A., Dugar S., Lachowicz J., et al.: Diphenylsulfone muscarinic antagonists: Piperidine derivatives with high M-2 selectivity and improved potency. Bioorg. Med. Chem. Lett. 10(19), 2209-2212, (2000). Rackley R., Weiss J.P., Rovner E.S., Wang J.T., Guan Z.: Nighttime dosing with tolterodine reduces overactive bladder-related nocturnal micturitions in patients with overactive bladder and nocturia. Urology 67(4), 731-736, (2006). Kaur K., Aeron S., Bruhaspathy M., Shetty S.J., Gupta S., Hedge L.H., Silamkoti A. D.V., Mehta A., et al.: Design, synthesis and activity of novel derivatives of oxybutynin and tolterodine. Bioorg. Med. Chem. Lett., 15(8), 2093-2096, (2005).
Ferreira et al. [64]
[65] [66] [67]
[68]
[69] [70] [71]
[72] [73]
[74]
[75]
[76]
[77]
[78]
[79]
[80]
[81]
[82]
[83]
[84]
[85]
[86]
[87]
[88]
Moisey C.U., Stephenson T.P., Brendler C.B.: The urodynamic and subjective results of treatment of detrusor instability with oxybutynin chloride. Br. J. Urol. 52(6), 472-475, (1980). Thompson, I.M., Lauvetz, R.: Oxybutynin in bladder spasm, neurogenic bladder, and enuresis. Urology, 8(5), 452-454, (1976). Anderson K.E., Chapple C.R.: Oxybutynin and the overactive bladder. World J. Urol. 19(5), 319-323, (2001). Yarker Y.E., Goa K.L., Fitton A.: Oxybutynin – a review of its pharmacodynamic and pharmacokinetic properties, and its therapeutic use in detrusor instability. Drugs Aging 6(3), 243-262, (1995). Starck J.P., Talaga P., Quéré L., Collart P., Christophe B., Brutto P.L., Janot S., Chimmanamada D., et al.: Potent anti-muscarinic activity in a novel series of quinuclidine derivatives. Bioorg. Med.Chem. Lett. 16(2), 373-377, (2006). Triggle D.J., Triggle C.J.: Chemical Pharmacology of The Synapse, Academic Press, New York, (1976). Dahlbom R., Ariens D.J.: Stereochemistry and Biological Activity of Drugs, Blackwell Scientific, Oxford, (1983). Angeli P., Cantalamessa F., Cavagna R., Conti P., De Amici M., De Micheli C., Gamba A., Marucci G.: Synthesis and pharmacological characterization of enantiomerically pure muscarinic agonists: Difluoromuscarines. J. Med. Chem. 40(7), 1099-1103, (1997). Dahanukar S.A., Kulkarni R.A., Rege N.N.: Pharmacology of medicinal plants and natural products. Ind. J. Pharmacol. 32(4), S81-S118, (2000). Lima Araújo K.G., Domingues J.R., Sabaa-Surur A.U.O., Silva A.J.R.: Production of antioxidants by Anabaena PCC7119 and verification of their protecting activity against oxidation of soybean oil. Food Biotechnology 20(1), 65-77, (2006). Grangeiro M.S., Calheiros-Lima A.P., Martins M.F., Arruda L.F., Garcezdo-Carmo L., Santos W.C.: Pharmacological effects of Eugenia punicifolia (Myrtaceae) in cholinergic nicotinic neurotransmission. J. Ethnopharmacol. 108(1), 26-30, (2006). Singh S., Kate B.N., Banerjee U.C.: Bioactive Compounds from Cyanobacteria and Microalgae: An Overview. Critic. Rev. Biotechnol. 25(3), 73-95, (2005). Sarada R., Manoj G., Pillai M.G., Ravishankar G.A.: Phycocyanin from Spirulina sp: Influence of processing of biomass on phycocyanin yield, analysis of efficacy of extraction methods and stability studies on phycocyanin. Proc. Biochem. 34(8), 795-801, (1999). Vadiraja B.B., Gaikwad N.W., Madyastha K.M.: Hepatoprotective effect of C-phycocyanin: Protection for carbon tetrachloride and R-(+)-pulegone mediated hepatotoxicity in rats. Biochem. Biophys. Res. Commun. 249(2), 428431, (1998). Romay C., Armesto J., Remirez D., Gonzalez R., Ledon N., Garcis I.: Antioxidant and antiinflammatory properties of C-phycocyanin from blue green algae. Inflamm. Res. 47(1), 36-41, (1998). Bhat V.B., Madyastha K.M.: C-phyocyanin: A potent peroxyl radical scavenger in vitro and in vivo. Biochem. Biophys. Res. Commun. 275(1), 20-25, (2000). Hsiao G., Chou P.H., Shen M.Y., Chou D.S., Lin C.H., Sheu J.R.: CPhycocyanin, a Very Potent and Novel Platelet Aggregation Inhibitor from Spirulina platenses. J. Agric. Food Chem. 53(20), 7734-7740, (2005). Bonnard I., Rolland M., Salmon J.M., Debiton E., Barthomeuf C., Banaigs B.: Total structure and inhibition of tumor cell proliferation of laxaphycins. J. Med. Chem. 50(6), 1266-1279, (2007). Becher P.G., Gademann K., Jüttner F.: Nostocarboline: isolation and synthesis of a new cholinesterase inhibitor from Nostoc 78-12A. J. Nat Prod. 68(12), 1793-1795, (2005). Cox P.A., Banack S.A., Murch S.J., Rasmussen U., Tien G., Bidigare R.R., Metcalf J.S., Morrison L.F., et al.: Diverse taxa of cyanobacteria produce N-methylamino-L-alanine, a neurotoxic amino acid. Proc. Natl. Acad. Sci. USA 102(14), 5074-5078, (2005). Cox P.A., Banack S.A., Murch S.J.: Biomagnification of cyanobacterial neurotoxins and neurodegenerative disease among the Chamorro people of Guam. Proc. Natl. Acad. Sci. USA 100(23), 13380-13383, (2003). Grover J.K., Vats V., Rathi S.S.: Anti-hyperglicemic effect of Eugenia jambolana and Tinospora cordifolia in experimental diabetes and their key metabolic enzymes involved in carbohydrate metabolism. J. Ethnopharmacol. 73(3), 461-470, (2000). Sharma, S.B., Nasir, A., Prabhu, K.M., Murthy, P.S., Dev, G.: Hypoglycaemic and hypolipidemic effect of ethanolic extract of seeds of Eugenia jambolana in alloxan-induced diabetic rabbits. J. Ethnopharmacol. 85(2-3), 201206, (2003). Ravi K., Sivagnanam K., Subramanian S.: Anti-diabetic activity of Eugenia jambolana seed kernels on streptozotocin-induced diabetic rats. J. Med. Food 7(2), 187-191, (2004). Consolini A.E., Baldini O.A.N., Amat A.G.: Pharmacological basis for the empirical use of Eugenia uniflora L. (Myrtaceae) as antihipertensive. J. Ethnopharmacol. 66(1), 33-39, (1999).