Chapter - I 1 1.1. Introduction to amino acids

Chapter - I
1.1. Introduction to amino acids
Although over 300 amino acids have been described in nature, only twenty of these are
typically found as components in human peptides. Of the twenty coded for by human
DNA, ten are considered essential since human metabolic pathways are insufficient to
synthesize them at adequate rates from other precursors. The essential amino acids are
arginine, histidine, methionine, threonine, valine, isoleucine, phenylalanine, tryptophan,
leucine, and lysine. However, arginine and histidine are essential only during periods of
high anabolic activity, such as tissue growth during childhood. Although amino acid
function is usually linked to peptide structure and function.
Free essential amino acids play a significant role in both primary metabolism and in the
formation of specialized nitrogen containing products. Some contribute to intermediates
of the Glycolytic and Krebs (Tricarboxylic Acid Cycle) cycles. Hence, amino acids are
used as a source of energy when carbohydrate or fatty acid levels have been depleted [1].
Some tissues, such as muscle, have the capacity to directly catabolize the branched chain
essential amino acids valine, leucine, and isoleucine [2]. Others play a part in
neurotransmitter formation and can alter significant physiological characteristics such as
mood. Tryptophan, for example, is a precursor of the neurotransmitter 5hydroxytryptamine (serotonin) [3]. Methionine is an example of an essential amino acid
that has a significant role in formation of special products requiring single carbon
transfers. Modifications occur to methionine which produces a high energy Sadenosylmethionine product. This metabolite has an important role in the formation of
choline, a significant part of many other high-energy metabolic intermediates [4]. There
is a tremendous amount of literature dealing with the physiological significance of free
Chapter - I
amino acids and their effects on a variety of metabolic and physiological systems. Their
importance is often illustrated when a nutritional deficiency occurs or an important
enzyme is defective.
Deficiencies in essential amino acid metabolism give rise to a plethora of pathological
conditions. The absence of an essential amino acid in the diet or a genetic defect in a key
enzyme can render other metabolites essential, such as phenylalanine [5]. Tyrosine is not
essential so long as an adequate supply of both phenylalanine and the necessary enzyme
to convert phenylalanine to tyrosine is present [6]. However, tyrosine becomes an
essential amino acid when a deficiency or genetic defect occurs in this system. Some
enzymatic deficiencies are benign, but others are debilitating and life-threatening. An
understanding of the pathways of essential amino acid metabolism is vital for effective
treatment of genetic defects altering these reactions. These defects provide insight into
the important roles essential amino acids have in human metabolism.
Amino acids and their metabolic and physiological ramifications are among the most
investigated topics in biomedical science. Many current studies focus on the therapeutic
applications of exogenous amino acids. The essential amino acid metabolism and
therapeutic modalities, the focus will be to describe the effects of exogenous amino acid
administration on each physiological system. Although amino acids most importantly
give rise to complex peptides such as immunoglobulins, carrier proteins, and
neurotransmitters, they also have unique biological and metabolic qualities as free amino
Chapter - I
1.1.1. Overview of the physical properties of the essential amino acids
Amino acids, as the building blocks of the most diverse biological compounds, have a
characteristic structure. With the exception of proline, all twenty amino acids have an
amino group and a carboxyl group with a functional group covalently bound to the alpha
carbon. In the essential amino acids, the functional groups are used to classify the amino
acids into polar, non-polar, or basic side chains. The chemical nature of the side chain
found on a particular amino acid plays a very important role in determining where the
amino acid is found in the tertiary structure of a peptide chain.
There are several ways to classify amino acids, the most prevalent being the chemical
nature of the side chains. Most essential amino acids have nonpolar functional groups.
These include valine, leucine, isoleucine, phenylalanine, tryptophan, and methionine. The
side chains in this category do not accept or donate protons (H+) at physiological pH.
Also, there is no participation in hydrogen bonding. Amino acids with nonpolar side
chains tend to be located on the interior of protein molecules where they interact with
other non polar (aliphatic) amino acids, and are shielded from polar entities, including
water [6]. One exception to this is proteins located in lipid rich environments like cellular
membranes, where a reverse arrangement occurs and the nonpolar amino acids are
located peripherally to the peptide [6].
Threonine has an uncharged polar side chain, which is neutral (uncharged) at the
physiological pH of 7.4. Threonine contains an uncharged polar hydroxyl group and is
capable of hydrogen bonding, which contributes to the tertiary structure of peptides. The
hydroxyl substituent can serve as an important site of attachment for other molecules
during biochemical reactions. Threonine can undergo a dehydration reaction with an
Chapter - I
oligosaccharide to form glycoproteins. The specific three-dimensional location of
threonine in a peptide is a determining factor as to whether or not the hydroxyl group will
be glycosylated. This type of reaction usually occurs as a peptide is being transferred
from the endoplasmic reticulum to the Golgi apparatus [7].
Histidine, lysine and arginine are classified according to their basic side chains. By
convention, these amino acids are proton acceptors at physiological pH (7.4). At this pH
of 7.4, lysine and arginine are protonated and positively charged. Histidine, however, has
a pKa of 6.0 for the imino nitrogen and is weakly basic. As a result, histidine in primarily
in an uncharged state as a free amino acid at physiological pH (7.4) [8]. Histidine may
have either a positive or neutral charge depending upon the microenvironment of the
component peptide it is a part of.
The pKa of amino acid side chains is a reliable predictor of which will be an effective
physiological buffer. Usually, a weak acid and its conjugate base will effectively buffer a
solution within ±1 pH unit of its pKa. For histidine, the pKa of the imidazole nitrogen in
the side chain is approximately 6.0 [8]. When the pH is lower than the pKa, the
protonated form is most abundant, and when pH is above pKa, the deprotonated form
predominates. This physical quality enables histidine to be a significant physiological
buffer. Although the most effective pH at which histidine can buffer a solution
corresponds to the pKa, 6.0 in this case, the concentration of the acid and conjugate base
are important as well. The more concentrated they are the greater the "buffering
Side chain characteristics, the ionic form present at a particular pH, and the acid/base
qualities of free amino acids help develop a picture of how these biological molecules
Chapter - I
function in their diverse roles. The nature of the histidine side chain explains how plasma
pH is protected from dangerous fluctuations. Reactivity of the hydroxyl component of the
threonine side chain illustrates that when oligosaccharides are added in a hydrolysis
reaction, the result is the formation of glycoproteins [5].
1.1.2. Metabolism of essential amino acids
The purpose of this section is to provide a general overview of the basic processes that
allow the essential amino acids to participate in intermediate metabolism as well as form
special products. Nutritional considerations
Essential amino acids must be obtained from the diet. A controversy currently exists as to
the appropriate dietary quantity for each of the essential amino acids. The debate is partly
due to inadequate previous technology for measuring amino acid requirements, such as
nitrogen balance and growth [9]. Most estimates of essential amino acid requirements,
based on nitrogen balance appear too low [9,10], with the exception of the sulfurcontaining methionine [11]. Other techniques used to estimate amino acid requirements
are: plasma amino acid concentration, direct amino acid oxidation and indicator amino
acid oxidation [9]. Protein metabolism in the body causes the elimination of nitrogen by
urea production, which is the quantitatively most important catabolite. The conversion of
the remaining amino acid carbon skeletons to intermediates of energy-producing
pathways and other intrinsic metabolic consumption creates a steady need for dietary
essential amino acids. The minimal nutritional requirement for humans and other
mammals is heavily effected by age, stage of growth and metabolic state. For example,
Chapter - I
children and patients in a catabolic state (i.e. burn, surgery) have a greater basal
metabolic requirement for the essential amino acids than does a healthy adult [10]. Digestion
In the American diet, the typical consumption of dietary amino acids is approximately
100 g/day [7]. These amino acids initially enter the body in the form of proteins and are
subsequently hydrolyzed by chemical reagents and enzymes. Most of the proteolytic
enzymes responsible for degrading dietary protein come from the stomach, pancreas and
small intestine [12].
Dietary protein digestion begins in the stomach as a result of gastric juice secretion.
Gastric juice contains both hydrochloric acid and pepsin. Although the stomach pH is not
sufficiently acidic to hydrolyze most peptides, it does denature them, making enzymes
more efficient at hydrolyzing the peptide bonds. Pepsin is secreted as the proenzyme
pepsinogen. Upon exposure to the acidic environment in the stomach, pepsinogen is
converted to the active form and is referred to as pepsin. Pepsin is unique in that other
active pepsin molecules can autocatalytically activate pepsinogen as well. Pepsin
cleavage of peptides releases a few free amino acids and smaller peptides which then
enter the duodenum of the small intestine [7].
Pancreatic proteases act in the small intestine to further degrade the peptides produced by
the action of pepsin in the stomach. The pancreatic proteases are also released as inactive
proenzymes which are all activated by the active form of trypsin, which is converted
from the inactive form, trypsinogen, by a brush border enzyme called enteropeptidase
[12]. All the pancreatic proteolytic enzymes have specificity regarding the peptide bonds
they cleave [12]. Details about the specific enzymes and amino acids cleaved are not
Chapter - I
essential to this overview of digestion. However, the end result is the production of
oligopeptides (smaller peptides) and additional free amino acids.
The final stage of peptide digestion involves aminopeptidase, an enzyme located at the
luminal border of the small intestine. This enzyme repeatedly cleaves the N-terminal
amino acid of the oligopeptides to produce free amino acids and some dipeptides [12].
Although both the free amino acids and dipeptides are absorbed into the epithelial cells of
the small intestine, only free amino acids are found in the portal system. While in the
cytosol of the intestinal epithelial cells the dipeptides are hydrolyzed to free amino acids.
After release into the portal system, the free amino acids are either metabolized by the
liver or released into the general circulation [12]. Removal of nitrogen and excretion
Since there is no storage form of dietary nitrogen, any nitrogen not immediately required
for the metabolic processes must be eliminated from the body. Transamination and
oxidative deamination reactions are responsible for the initial step of amino acid
catabolism. The alpha-amino nitrogen is removed by either of these processes [13]. With
transamination, there is the production of an alpha-ketoacid and glutamate. Of the
essential amino acids, only lysine does not undergo a transamination [7]. The alphaketoacids may participate in the synthesis of other non essential amino acids, while the
glutamate formed can be deaminated to form alpha ketoglutarate. Alpha-ketoglutarate is
the receptor of the alphaamino group in transamination reactions [13].
Oxidative deamination results in the direct elimination of the alpha-amino group as
ammonia. The ammonia produced in this reaction acts as a source of nitrogen in urea
production. None of the essential amino acids undergo rapid oxidative deamination.
Chapter - I
However, the glutamate formed by transamination is converted to ammonia by the
glutamate dehydrogenase enzyme. This indirect mechanism provides a method for
converting the amino group of most amino acids to ammonia. In this way, glutamate acts
as a funnel by which many amino acids can be converted to intermediates of the TCA
cycle [5].
The ammonia produced by oxidative deamination provides a source of nitrogen in the
Urea Cycle, which is the major pathway for elimination of amino acid nitrogen.
Production of urea occurs in the liver and is excreted in the urine [5].
1.1.3. Special products and systemic effects of essential amino acid metabolism
Aside from their important roles in enzymes and other proteins, essential amino acids are
also precursors for many physiologically significant nitrogencontaining molecules.
Among the special products are; neurotransmitters, hormones, and nucleotide bases. This
section describes briefly the roles of essential amino acids in the formation of these and
other physiologically significant products. Arginine
During catabolism, arginine is cleaved by arginase to produce ornithine which is then
transaminated to yield glutamate semialdehyde and is subsequently converted to alpha
ketoglutarate via glutamate. Arginine can be converted to creatine when combined with
glycine and a methyl group from Sadenosylmethionine. Creatine is reversibly
phosphorylated to creatine phosphate, which is a high energy derivative that reversibly
donates a phosphate group to ADP in the formation of ATP. This reaction provides a
small but rapidly mobilized energy reserve in muscle [8]. In the liver arginine serves an
important role in the excretion of nitrogen containing compounds. As an intermediate in
Chapter - I
the urea cycle, arginine is cleaved into ornithine and urea. The urea is excreted in the
urine while the ornithine reenters the urea cycle [5]. The arginine is the primary
component in the synthesis of nitric oxide (NO) [14]. Nitric oxide is synthesized from
arginine, molecular oxygen, and NADPH by the enzyme nitric oxide synthase.
Arginine, through nitric oxide (NO), is a mediator in a variety of biological systems.
Since nitric oxide is functionally identical to endothelium-derived relaxing factor, it
causes vasodilation by relaxing vascular smooth muscle [15,16,17]. Studies with
mineralocorticoid-salt hypertensive rats and corticotropininduced high blood pressure
have found that L-arginine has an antihypertensive effect. The mechanisms of action for
arginine are through the vascular effects of NO and the reduction of serum corticosterone
concentrations respectively [15,17]. In addition, arginine's inhibition of the reninangiotensin system helps to cause a decrease in the systolic blood pressure [18].
In a study of healthy men it was found that oral administration of L-arginine prevented
platelet aggregation. Its inhibition was directly correlated to plasma levels of arginine,
while heart rate and fasting lipid levels were unaffected [19]. Another effect of arginine,
D- and L-stereoisomers, is the production of hormonal responses [20]. Systemic infusion
of arginine increases the plasma insulin, glucagon, and prolactin levels [21]. Also,
arginine, through nitric oxide (NO), stimulates the release of dopamine from the striatum
in gerbils [22], increases the tumoricidal and bactericidal actions of macrophages [23],
stimulates wound healing [24], and has neurotransmitter functions in the brain [25]. Tryptophan
Tryptophan is converted into two products: niacin and the neurotransmitter serotonin
[26]. Serotonin has many diverse physiological functions such as pain perception [27,28],
Chapter - I
affective disorders and sleep [29], temperature [30] and blood pressure [31]. In the
formation of serotonin, tryptophan is converted to 5-hydroxytryptophan in which
tetrahydrobiopterin and molecular oxygen are necessary for this reaction. 5hydroxytryptophan is then decarboxylated to serotonin. Although niacin is readily
available from lean meat, cereals and enriched grains, it can also be synthesized
inefficiently from tryptophan [26]. The metabolic pathway producing niacin from
tryptophan only produces 1 mg of nicotinic acid for every 60 mg of tryptophan
consumed. The production of niacin is only functional when a relative overabundance of
the amino acid is available. This in vivo inefficiency renders dietary niacin as the primary
source [26].
An increase in tryptophan levels leads to an increased concentration of 5hydroxytryptamine (serotonin) in the brain. This effect can be observed in neurons which
are related to sleep function [29]. An excess of serotonin in brain tissue is known to
increase the mental effort necessary to maintain physical exercise [1,32]. Acute ethanol
consumption is known to decrease circulating tryptophan availability [33]. One of the
major systemic effects of tryptophan is through its 5-hydroxytryptamine metabolite [7].
Serotonin has been implicated in various psychiatric disorders [34]. Tryptophan depletion
has been used successfully to evaluate central serotonin levels in depression and other
neuropsychiatric disorders. Tryptophan depletion reduces brain serotonin function, which
in turn reduces the therapeutic effects of specific serotonin reuptake inhibitors, but not
drugs that inhibit noradrenaline reuptake. Rather than having a neurotransmitter function,
tryptamine has been suggested to play a neuromodulatory role. A decrease in erythrocyte
tryptophan uptake in schizophrenic patients is correlated with a loss of impulse control
Chapter - I
[35]. The decrease in tryptophan uptake is a reflection of peripheral tryptophan
metabolism. The majority of scientific literature indicates that 5-hydroxytryptamine acts
directly as a neurotransmitter.
Tryptophan has been implicated as a possible explanation of central fatigue [1,32].
During extended exercise free tryptophan levels are elevated and, therefore, serotonin [1].
This increases activity in the brain of neurons that can induce sleep. Phenylalanine
Pietz et al. found that when phenylketonuria (PKU) patients were administered a bolus of
100 mg/kg phenylalanine, both the plasma and brain concentrations of phenylalanine
increased significantly with no impairment of attentional and fine motor scores on
neurophysiological tests for up to 20 hours postload [36]. However, most of the systemic
effects of phenylalanine are a result of the enzyme phenylalanine hydroxylase converting
phenylalanine to tyrosine [37]. Thus, this nonessential amino acid tyrosine overlaps
substantially with phenylalanine. L-tyrosine is ultimately converted to fumarate and
acetoacetate intermediates which join primary metabolic pathways as both glucogenic
and ketogenic precursors [6]. Phenylalanine and tyrosine form the initial two biosynthetic
steps of dopamine formation [38]. The rate limiting enzyme that catalyzes dopa, the
immediate precursor to dopamine, is tyrosine hydroxylase [39]. This enzyme is abundant
in the adrenal medulla, sympathetic nervous ganglia and in the central nervous system
[40,41]. Dopamine serves as a neurotransmitter in the brain whose importance is
illustrated by Parkinson's disease where nuclei in the subthalmic region fail to produce
adequate quantities of this neurotransmitter [42]. Deficiency of phenylalanine causes a
Chapter - I
diminished synthesis of dopamine which has adverse effects on performance of mental
tasks [37].
Dopamine, a precursor of epinephrine and norepinephrine, when hydroxylated forms
norepinephrine [43]. Norepinephrine is a catecholamine that is released as a
neurotransmitter from postganglionic sympathetic nerves and also functions as a
Sadenosylmethionine, a metabolite of the essential amino acid methionine, donates a
methyl group to norepinephrine to form epinephrine (i.e. adrenalin) [44]. Histidine
Histidase deaminates histidine to form N-formiminoglutamate (FIGlu), which transfers
the formimino substituent to tetrahydrofolate and forms glutamate [5]. A dietary
deficiency of folic acid results in increased urinary elimination of FIGlu and is the basis
of the FIGlu excretion test [45,46]. Tetrahydrofolic acid (THF), a metabolite of histidine
and the active form of folic acid, serves an important role in one carbon metabolism.
Dihydrofolate reductase converts the inactive folic acid to its active form with the
addition of two protons (H+) and two molecules of NADPH. The various forms of THF
are essential in methionine, thymidine, purine-C8, and purine-C2 synthesis. Unlike
humans, some microorganinsms synthesize folic acid directly. In humans, methotrexate
inhibits the conversion of folic acid to its active form and is a useful regimen for patients
with acute lymphocytic leukemia [47,48].
Histidine has effects on a variety of organisms and physiological systems. As a free
radical scavenger, histidine quenches hydroxyl and hydrogen peroxide, but not
Chapter - I
superoxide anions [49]. The total free energy of cells is increased as the adenine
nucleotide pool is elevated following histidine administration [49].
Histidine plays an important role in the modulation of oxidative DNA degradation.
Protonation of the imidazole component of histidine abolishes the capacity of histidine to
modulate the oxidative degradation of DNA [50]. Evidence suggests that at low hydrogen
peroxide levels, the protective effect of histidine may be a result of its ability to bind
hydroxyl free radicals. Histidine also reduces ischemia induced by myocardial injury in
isolated perfused rat hearts [49].
An important metabolite of histidine is the chemical messenger histamine. Histidine is
decarboxylated to form this powerful component of many allergic and inflammatory
reactions. Histamine mediates a variety of cellular actions such as gastric acid secretion
[51], vasodilation [52], and allergic reactions [53]. Histamine has also been suggested as
a possible neurotransmitter in the brain [53]. Although histamine is not used clinically,
histamine antagonists have important therapeutic functions. Branched chain amino acids (valine, leucine, isoleucine)
The branched chain essential amino acids (BCAA) have similar characteristics, properties
and physiological actions. These amino acids are unique metabolically in that they are
primarily catabolized by peripheral tissues (i.e. skeletal muscle) and have similar
catabolic pathways [2]. They are first transaminated to alpha ketoacids by the enzyme,
branched chain alpha-ketoacid dehydrogenase. This step requires the aid of several
coenzymes, thiamine pyrophosphate being the most prevalent. The end result is succinyl
CoA for valine, acetyl CoA for leucine (the second of two exclusively ketogenic amino
Chapter - I
acids) and succinyl CoA or acetyl CoA for leucine and succinyl CoA or acetyl CoA for
isoleucine [2].
BCAA have a protein sparing effect when their levels are increased through infusion or
oral administration. An overnight infusion of BCAA was associated with a 20-60%
decline in arterial concentrations of the other amino acids. This suggested that the BCAA
inhibit proteolysis in skeletal muscle and other body tissues [54,55]. In addition to the
protein sparing effects, leucine and the other BCAA exert their effects on respiratory
functions. Investigations have shown that respiratory drive increased and diaphragm
function improved when BCAA were administered [56]. Increased levels of BCAA have
been shown to decrease pCO2 and to stimulate the ventilatory response to hypercapnia.
Also, a decrease in episodes of apnea in premature infants was found with an increase in
BCAA [57]. Leucine metabolism has demonstrated an effect in the brain. Astrocytes in
the brain metabolize leucine and its nitrogen furnishes the alpha amino group for
glutamate synthesis. Glutamate sequesters free ammonia in the brain which is very
sensitive to hyperammonemia. During this process, glutamate is converted to glutamine
[58,59]. This is an important step in the brain because glutamate functions as a
neurotransmitter, regulates ammonia levels, serves as a constituent for glutathione and
folic acid and serves as a precursor of gammaamino butyric acid (GABA) and other
Krebs Cycle intermediates [58,59]. However, glutamate is unable to cross the blood brain
barrier. Hence, leucine levels are significant in the overall nitrogen metabolism in the
Chapter - I Methionine
Methionine undergoes an important metabolic reaction when it is converted to S
adenosylmethionine (SAM). SAM is the primary methyl group donor in one carbon
metabolism [2]. In SAM synthesis, L-methionine is coupled with ATP by the enzyme Sadenosylmethionine synthase with magnesium as a cofactor [8]. This produces the high
energy SAM, which is unusual in that it contains no high energy phosphate bonds. All
three high energy phosphate bonds in ATP are cleaved during the synthesis of SAM. The
methyl group attached to the tertiary sulfur in SAM is irreversibly transferred to other
molecules in a step catalyzed by methyltransferases.
Subsequent to transfer of the one carbon unit, SAM is hydrolyzed to homocysteine and
adenosine by the addition of water to one of the sulfur bonds [8]. Homocysteine reacts
with serine through cystathione synthase to form cystathione. Where as cysteine and
alphaketobutyrate are oxidated to form propionyl CoA and then subsequently succinyl
CoA. The metabolism of valine, isoleucine, threonine, and methionine converge at
succinyl CoA to enter the Krebs Cycle [2]. Methionine is another example of an amino
acid that is not essential as long as its essential precursor is present. That is, a decrease in
methionine will cause a deficiency in cysteine [7]. Homocysteine may also be used in the
resynthesis of methionine, the initial precursor in the pathway. N5-methyl-THF donates a
methyl group to homocysteine in a reaction requiring methylcobalamin. This results in
the resynthesis of methionine and production of tetrahydrofolate. Methylcobalamin, the
coenzyme, is a metabolite of vitamin B12 [2].
Methionine is also an important factor in the synthesis of choline [4]. Synthesis of
choline requires the addition of three methyl groups from the activated form or SAM.
Chapter - I
Methionine deficiency causes an insufficient supply of choline even though choline can
be resynthesized from phosphatidylserine in membranes [4]. Lysine
The catabolism of lysine results in the formation of acetoacetate. It is unique in that
neither of its amino groups undergo transamination as the first step in their catabolism. In
the mammalian liver, lysine first forms alphaaminoadipate-delta semialdehyde before it is
converted to acetoacetate [5]. In rats, excess dietary lysine has a significant effect upon
the distribution of carnitine and trimethyllysine (TML), a carnitine precursor. In a high
lysine diet, plasma concentration of carnitine is decreased while the concentrations of
trimethyllysine are increased in the plasma and in skeletal muscle [60]. TML has been
shown to stimulate cell proliferation in bone marrow, intestinal tissues and in cultured
lymphocytes [61]. L-lysine has also been shown to depress the central nervous system
[3H]flunitrazepam binding in the brain [62]. Threonine
When catabolized, threonine is dehydrated first to alpha-ketobutyrate, which is then
converted to propionyl CoA, the precursor of succinyl CoA. Through threonine
metabolism pyruvate is formed, which also enters the Krebs cycle [5]. In diet-induced
hyperthreoninemia, increased quantities of threonine and glycine were found in brain
tissue. When moderate levels were administered, glycine did not increase in the brain. It
was discovered that glycine was increased only at high levels of dietary threonine [63]. In
the peripheral tissues, increased glycine concentrations again resulted from a high
threonine diet. Hepatic threonine dehydrogenase activity was induced in these studies
Chapter - I
[64]. Rats fed a threonine imbalanced diet exhibited altered hepatic metabolism of longchain free fatty acids [65]. There was a 2-4 fold increase in triglyceride levels as well as
enlarged livers. Excess threonine may be a causative factor in hypertension. Rats given
and 8% threonine solution exhibited an elevated systolic blood pressure and a thickening
of their small arterial walls [66].
1.1.4. Overview of the therapeutic effects of the essential amino acids
The therapeutic application of essential amino acids has received considerable attention
in respiratory physiology, cardiology, renal failure, neurological disorders, and congenital
defects. The benefits of exogenous essential amino acid therapy lie in the relative
abundance and economy of these biologically active materials. This section deals with an
overview of essential amino acid therapeutic applications. Methionine
As discussed previously, methionine, a sulfur containing amino acid, is also very
important in single-carbon metabolism in the activated SAM form. Methionine also plays
a role in detoxification in the liver [67,68]. In chronic alcoholics, methionine metabolism
is significantly altered [68,69], producing a deficiency of SAM. This deficiency,
observed in cirrhotic subjects, is located at the Sadenosylmethionine synthase step in
methionine metabolism [70]. Investigators have considered the feasibility of giving
exogenous doses of the methionine derivative, SAM to ameliorate many of the
complications observed in chronic liver diseases and cirrhosis [68,70]. Through
therapeutic doses of SAM, glutathione would also be increased, leading to protection
against oxidant stress from druginduced hepatotoxicity [70,71]. Preliminary studies
regarding this application have given promising results in both the improvement in liver
Chapter - I
function tests and biochemical parameters of choleostasis [70]. Dietary supplementation
of methionine also increases levels of other free-radical scavengers and lowers the
succeptability to lipid peroxidation [72]. Jaundiced patients showed a decrease in
extrahepatic membrane cholesterol deposition due to exogenous SAM administration
[73]. However, some liver pathology, such as patients with intrahepatic choleostasis, has
no response to SAM administration [74].
Amino acids have been known to enhance the pharmacological effects of some other
drugs when given in together [75]. One such application of methionine has been observed
in animal experiments involving lead intoxication in rats [76]. Ethylenediamine tetra
acetic acid is a chelating agent used clinically in patients with heavy metal poisoning.
Supplementation of this regimen with zinc and methionine increased urinary excretion of
lead. However it did not reverse the lead-induced biochemical alterations [76].
Therapeutically, methionine may be used clinically as a prophylactic to guard against
congenital defects from teratogen exposure. Exogenous methionine has been shown to
reverse the teratogenic effect of trans-retinoic acid in mice [77].
Some cancer cells displayed a methionine-dependant growth pattern [78,79]. The
relationship between methionine dependency and the metastatic potential of a rat cancer
line has been demonstrated experimentally. The greater the metastatic potential, the
greater the concentration of methionine required to maintain growth [78]. That is,
methionine dependent cancer cell lines lost their tumorigenicity when they were injected
into rats fed a methioninedeprived diet. However, this special diet substituted
homocysteine for methionine to maintain growth of normal cells. Exploiting this
metabolic defect in the cancer cell lines may be of possible therapeutic value [78].
Chapter - I
Similar anticarcinogenic effects were observed in other studies [80]. Also the reestablishment of physiological SAM levels in rats has been shown to inhibit
protooncogene expression and prevent lesion development in vivo [81].
A derivative of methionine, methionine sulfoxamine, reduces cortical infarct size in rats
after middle cerebral artery occlusion [82]. The mean volume of the infarct in the cortex
was reduced by 33 % in the group treated with the sulfoxamine analogue. Sulfoxamine
also enhances brain glycogen stores [82]. This finding could provide a new therapeutic
approach for stroke patients. Tryptophan
Tryptophan is the precursor of 5-hydroxy-tryptophan (serotonin), a potent effector of
mood and behavior as well as a neurotransmitter or neuromodulator [83]. Peripheral
tryptophan metabolism directly affects plasma tryptophan availability and consequently
serotonin synthesis [35]. Therefore, tryptophan levels have an effect on depression and
mood. Serotonin, a monoamine, is related to the catecholamine hormones norepinephrine
and epinephrine. The therapeutic applications of tryptophan and its tryptamine metabolite
are dependent on their physiological actions of regulating mood, sleep, motor activity,
thermoregulation, sexual activity, aggression, feeding, learning, and memory. Hence,
tryptophan is a highly significant biological molecule [84].
The determinations of tryptophan and serotonin concentrations in plasma have been a
valuable diagnostic aid in the study of drugs that alter serotonin metabolism [85,86]. One
of the main therapeutic applications for tryptophan is its enhancement of monoamine
oxidase inhibitors in the treatment of depression [75]. Tryptophan levels are one of the
most frequently used end-points in diagnostic and neuropsychopharmacological studies
Chapter - I
of serotonin function [87]. Tryptophan depletion, which reduces brain serotonin function,
is known to reverse the pharmacological action of particular serotonin reuptake inhibitors
(SRI), but not drugs that inhibit norepinephrine reuptake [86,88]. Examples of drugs in
this SRI category which are altered by reduced tryptophan levels include paroxetine and
clomipramine [88]. Rapid depletion of tryptophan in the diets of untreated depressed
patients did not result in an immediate change of mood, but did increase serotonin levels
on the day after the depletion test [89]. Other investigators reported little change in
depressed mood upon tryptophan depletion in drug-free depressed and healthy subjects
[86]. This suggests that reduced serotonin levels does not linearly relate to depression,
but may have a predisposing effect.
Pathological and immunological effects have been reported from exogenous L-tryptophan
administration. Eosinophilia-myalgia syndrome (EMS), associated with the ingestion of
exogenous tryptophan, is characterized by myalgia, eosinophilia, chronic cutaneous
lesions, progressive neuropathy, and myopathy [90,91,92]. Ingestion of exogenous Ltryptophan has also been found to induce pancreatic atrophy [93]. L-tryptophan, along
with other amino acids, has been shown to induce cholecystokinin (CCK) production and
subsequent pancreatic enzyme production in dogs and humans [94]. Tryptophan is the
most potent amino acid for stimulating pancreatic synthesis activity in dogs. Other
studies have implicated tryptophan and its metabolites in fibrosing illnesses like carcinoid
syndrome, eosinophilic fasciitis and scleroderma [95,96,97,98]. Although tryptophan
may have therapeutic applications in combination with antidepressants, other therapeutic
uses should be considered with caution due to the possible deleterious effects observed
from exogenous tryptophan ingestion.
Chapter - I Branched-chain amino acids
Valine, leucine and isoleucine are similar in both their metabolism and therapeutic
applications. Neurophysiological therapeutic effects have been found with branched
chain amino acid (BCAA) administration. Patients who suffered from chronic liver
failure or portal circulation defects showed a variety of neurological symptoms due to an
increase in nitrogenous ammonia in the systemic circulation. This condition is commonly
referred to as hepatic encephalopathy and is characterized by mood or personality
changes, drowsiness, coma, dysphasia, and asterixis [99]. Clinical trials have explored the
psychotropic effects of the BCAA and their antagonistic action in encephalopathy [99].
Neurophysiological improvements have been confirmed in quantitative psychometric
tests [100,101].
Dietary restriction of phenylalanine is essential in preventing brain damage in
(phenylketonuria) PKU patients [102]. BCAA have been used to treat phenylketonuria by
inhibiting the entry of phenylalanine into the brain [102]. Thus, there is a reduction of
central nervous systems (CNS) toxic effects caused by phenylalanine. The data from
clinical experimentation has shown that BCAA are useful and effective in maintaining
low serum phenylalanine levels [102]. Since BCAA have an inhibitory effect on
proteolysis, they have also been used as a treatment in septic patients and those with
catabolic disorders [103]. Leucine has a metabolic application in the determination of
protein turnover rate, where patients are primed with isotopes of leucine [104]. A
synthetic analog of leucine is successfully used in the clinical treatment of duodenal
peptic ulcer hemorrhage [105]. Patients in this study were administered a 3 mg dose
Chapter - I
daily. Only 8% of the subjects failed to respond to this treatment while 32% had a
positive result.
Isoleucine has a very low toxicity at pharmatological levels up to 8% solution
concentration in rats. Body weight, hematology and food consumption by rats were not
altered, although an increase in urine output and relative kidney size was observed [106].
Conversely, when BCAA were eliminated from diets in the treatment of Maple Syrup
Urine Disease, increased proteolysis was observed with isoleucine deficiency [107]. This
suggests that there is a risk when specific amino acids are eliminated from the diet
without adequate supplementation with other intermediates.
Besides phenylalanine, BCAA have an inhibitory effect on the transport mechanism of
other amino acids. Valine inhibits the transport of tryptophan across the blood-brain
barrier [108]. Since tryptophan is the precursor of 5-hydroxytryptamine (serotonin), a
mild decrease in brain serotonin neurotransmission was observed with subsequent
lowering of mood [108].
Valine deficient diets were shown to increase calcium excretion in urine with a reduction
in bone mass in chicks [109]. BCAA deficient diets have been studied for possible use in
anti-tumor therapy [110]. These investigators have demonstrated that valine and leucine
deficient diets had the most desirable effect in decreasing tumor growth with minimal
loss of body mass. However, diets deficient in all three BCAA had less effect on
inhibiting tumor growth and negatively impacted host development. This suggests that
selective removal of amino acids from the diet would be more beneficial in tumor therapy
with less weight loss than elimination of all three BCAA. Vitamin B12 deficiency causes
neurological deterioration if left untreated. The administration of valine and isoleucine,
Chapter - I
two precursors in the propionic acid pathway, protected against neurological damage in a
study group of B12 deficient bats [111]. It appears in this study that valine and isoleucine
circumvented this by stimulating the propionic acid pathway. Arginine
The therapeutic applications of arginine are numerous systemically. As previously
discussed, arginine is converted to nitric oxide (NO) which acts as a mediator in
vasodilation [112], congestive heart failure [113], inflammatory response [114],
chemotherapy [115], inflammatory pulmonary disease [116], pulmonary hypertension
[117], and axon growth [118]. Arginine reduces postischemic injury in the heart and
exerts antihypertensive and antiproliferative effects on vascular smooth muscle [16].
Arginine also prevents local vasospasms, unwanted proliferation of smooth muscle cells
and helps to control blood coagulation [119]. The nitric oxide synthase pathway in which
arginine is a precursor has been implicated in the pathogenesis of septic shock,
hypertension and atherosclerosis [120]. Studies in dogs have shown that a cardioplegic
solution supplemented with L-arginine reduces the infarct size, preserves postischemic
systolic and diastolic regional function and prevents arterial endothelial dysfunction
[121]. Without L-arginine supplementation, ischemic damage and contractile dysfunction
remained [121]. This study suggests that L-arginine may be a possible treatment in heart
attack patients. Capillary reperfusion with exogenous arginine after ischemic conditions
in hamsters further illustrates its effect on vascular function [122].
Arginine has been used in the treatment of necrotic colitis, which is caused by an
enterotoxin whose symptoms exhibit an infarction of the mucosa, edema and hemorrhage
[99]. The neuroeffector action causes smooth muscle relaxation, while nitric oxide
Chapter - I
maintenance of the intestinal mucosa protects the gut from blood-born toxins and tissue
destructive mediators. Thus, L-arginine may be considered as a potential therapy for
necrotic enterocolitis [23]. Arginine supplementation has also been used in the prevention
and treatment of osteoporosis. When pharmacological doses of arginine are administered,
growth hormone, IGF-1 and NO responses are induced [123]. Both GH and IGF-1 are
important mediators of bone deposition [124]. Therefore, L-arginine administration may
be part of an effective treatment to increase bone mass.
Arginine is also involved in the inflammation, tissue repair and fibrogenesis processes in
the kidney as described by [125]. Arginine is essential for the synthesis of several
metabolites that are secreted in the kidney such as creatine, urea and nitric oxide which is
excreted as nitrates and nitrites [126]. Dietary supplementation of arginine has resulted in
improvements of several kidney pathologies. These pathologies include subtotal
nephrectomy, diabetic nephropathy, cyclosporine A administration, salt-sensitive
hypertension, uretal obstruction, puromycine amino nucleoside nephrosis, kidney
hypertrophy due to high-protein diets and glomerular thrombosis [126]. Lysine
Lysine has been used in the treatment of recurrent herpetic lesions [127]. The herpes
simplex virus requires high concentrations of arginine to synthesize proteins and
replicate. Lysine acts as an anglogue of arginine and competes at the site of absorption in
the small intestine. Consequently, lysine prevents the development of the herpes labialis
lesions. Additionally, lysine is effective in the treatment of recurrent aphthous ulcers
Chapter - I
Lysine acetylsalicilate (lysine aspirin) and bendazac lysine (a lysine salt), both
derivatives of lysine, have been investigated for their therapeutic effects on migraine
headaches [128], acute respiratory infections [129] and rheumatoid arthritis [130]. Given
in combination with metoclopramide, lysine aspirin proved to be an effective treatment
for migraine headaches [128]. In the treatment of acute respiratory infections (laryngitis,
tracheitis, bronchitis, pneumonia) the effects of lysine aspirin was comparable to that of
nimesulide [129]. However, patients did incur more gastrointestinal side effects [129].
When comparing lysine aspirin with ibuprofen for the relief of rheumatoid arthritic
symptoms, more side effects were found with the lysine aspirin [130]. However, the
majority of patients preferred the lysine aspirin therapy over ibuprofen since there was
improved mobility and the alleviation of pain [130]. The bendazac derivative of lysine
may be useful in delaying the progression of cataract formation since it inhibites the
denaturation of proteins [131].
It has been reported that L-lysine supplementation can significantly enhance intestinal
calcium absorption and improve renal resorption of filtered calcium [132,133]. Since agerelated bone loss is due to calcium deficiency, L-lysine may prove to be an effective
treatment modality. Histidine
Histidine is a precursor of histamine, the mediator of many inflammatory and allergic
responses [7]. Histidine also has oxygen free radical scavenging qualities. An
investigation of the antioxidative properties of histidine in myocardial injury in rat hearts
showed that histidine prevented postischemic reperfusion injury [128]. Hearts treated
with histidine showed greater functional recovery with increased high-energy phosphates.
Chapter - I
Histidine was shown to quench hydroxyl radicals and hydrogen peroxide, but not
superoxide anions [49]. Another study involving subarachnoid hemorrhage in rabbits
showed that histidine attenuated cerebral vasospasm [134]. In a study on the effects of
histidine on brain edema and cardiac function after thrombotic ischemia in rats, it was
found that diminished histidine decreased brain water content and enhanced left
ventricular function in animals [135].
Histidine-containing dipeptides, carnosine and anserine, had an anti-inflammatory effects
and may be valuable in the wound healing process [136]. It is believed that these
dipeptides interact with oxygen radicals and lipid peroxidation products to prevent
membrane damage. Phenylalanine
Phenylalanine and its metabolite tyrosine are involved in the initial metabolic steps in
dopamine synthesis. An abundance of research has focused on the modulation of
phenylalanine in patients with phenylalanine hydroxylase deficiency [36,38,137]. As a
result of this genetic aberration, clinical literature concerning phenylalanine is involved in
the manipulation and metabolism of phenylalanine hydroxylase deficiency patients [138].
Analysis of nucleic material coding for the phenylalanine hydroxylase gene has been of
some benefit in predicting the outcome and severity of phenylketonuria (PKU) in humans
[139,140]. Some success has been achieved in correcting the phenylalanine hydroxylase
genetic deficiency through transduction of mice hepatocytes in vitro to produce
dramatically higher levels of phenylalanine hydroxylase [141]. Another genetic
aberration in phenylalanine metabolism has been elucidated in its predisposition for
Chapter - I
hypertension and stroke, which might be related to excessive stimulation of the
sympathetic nervous system [142].
A derivative of phenylalanine, 4-borono-2-[18F] fluoro-D, L-phenylalanine has shown
promise as a tracer in positron emission tomography (PET) for imaging of cancer cells
[143,144]. Phenylalanine has also been used diagnostically to determine protein synthesis
in visceral tissue [145]. This method of measuring protein synthesis represents an
improvement over continuous infusion methods in visceral tissues.
Cirrhosis of the liver is associated with elevated phenylalanine levels due to increased
proteolysis and decreased splanchnic extraction of dietary phenylalanine [146]. In
Cirrhosis, the ratio of branched-chain amino acids to phenylalanine is altered although
the mechanism for this is not completely understood [147]. The phenylalanine
metabolites, phenylethylamine and phenylacetic acid, are involved in encephalopathy in
sepsis and hepatic failure [148]. Threonine
Therapeutically, threonine has been used as a treatment for spinal spasticity and multiple
sclerosis [149,150]. Threonine enhances the glycinergic postsynaptic inhibition of the
motor reflex arc in the spinal cord with no toxic or adverse effects. When compared with
commonly used antispastic drugs, which commonly cause sedation and increased motor
wakness, threonine appears to be an appealing alternative [149]. In Amyotrophic Lateral
Sclerosis, a fatal neurological disease with no known cure [150], patients administered
threonine complained less frequently about disease-related respiratory failure [151].
Although threonine consumption at levels of four times that normally found in the diet of
rats did not impair their behavioral development [152], threonine metabolism may have a
Chapter - I
role in mediating hypertension. Specifically, ethanol and threonine are precursors of
acetaldehyde. Upon long-term dietary supplementation of threonine in rats, smooth
muscle cell hyperplasia increased while luminal diameters of small arteries and arterioles
decreased [66]. Thus it is apparent that acetaldehyde may be is implicated as the
mediating factor in both ethanol and threonine induced hypertension.
The ten essential amino acids are responsible for a vast array of metabolic, physiologic,
and therapeutic effects throughout the body. In addition to their roles in peptide and
protein structure, these free amino acids have significant functions as specialized nitrogen
containing products, neurotransmitters and as alternate energy sources via the Krebs
Cycle. Unique therapeutic uses of these biologically significant molecules are currently
being explored and could become an economic alternative to more expensive clinical
approaches. Since these biologically active amino acids must be obtained from the diet,
an over abundance or deficiency in just one of these may have severe pathological
consequences. Even though the metabolic, physiologic and therapeutic effects of these
essential amino acids have been extensively explored in some areas, they still remain an
important modality in clinical medicine. The negative systemic effects observed with
pharmacological doses of some amino acids, such as tryptophan, may limit their use
clinically. However, the diverse physiological and metabolic applications of many of the
essential amino acids will certainly yield a significant body of cost-effective alternative
therapeutic applications.
1.2. Introduction to peptides
Numerous small and large peptides, which are sequence and length-specific polymers
composed of amino acids, represent compounds with significant therapeutic applications.
Chapter - I
Peptides and their higher relatives proteins play a crucial role in almost all processes of
neurotransmitters, neuromodulators and hormones peptides are responsible for the
regulation of biochemical processes in complex organisms such as cell-cell
communication and control of vital functions like metabolism, immune response,
digestion, respiration, sensitivity to pain, reproduction, behaviour and electrolyte levels.
Since so many peptides possess potent pharmacological properties they are of enormous
medicinal interest.
Peptides are defined as polypeptide chains of less than 50 amino acids or 5000 Da
molecular weight, which often exhibit a high degree of secondary structure and lack
tertiary structure. Peptide leads have traditionally been derived from three sources:
isolated from nature (also known as bioactive peptides); chemical libraries or
genetic/recombinant libraries. To date, nearly all peptide therapeutics have been derived
from natural sources. One explanation is that bioactive peptides have undergone natural
selection and, as a result, have enhanced in vivo stability [153]. Bioactive peptides are
also highly functional with many serving as potent agonists and antagonists against
several receptors involved in disease progression. Some well-known bioactive peptides
include glucagon-like peptide-1 (GLP-1) for the control of diabetes, ghrelin to treat
obesity, gastrin-releasing peptide used in cancer treatments, and defensin, which has
found use as an antimicrobial agent. In the past, stable and potent peptides have been
difficult to discover from chemical and genetic/ recombinant peptide libraries. With the
Chapter - I
creation of highly diverse structured peptide libraries, however, peptides can now be
found that rival those found in nature.
Although peptides have several advantages over small molecules (e.g. higher
affinity/specificity to target and lower toxicity profiles) and antibodies (e.g. room
temperature storage and better tissue penetration owing to their smaller size), they have
been hampered in the past by several key issues that have prevented them from becoming
a mainstream source of drug candidates [154].
The short half-life of peptides has been one of the major issues of peptide therapeutics.
Peptides are typically cleared from the bloodstream within minutes to hours after
administration. As a result, there can be insufficient exposure in the target tissue to have
an in vivo effect. Short peptide half-lives typically result from fast renal clearance (for
peptides <5 kDa) and/or from enzymatic digestion in the blood, kidneys or liver. To
address this issue, numerous technologies have been developed to increase the in vivo
plasma residence time of peptides. These approaches have reached wide acceptance and
should help to appease peptide skeptics.
Peptide-based drug candidates that target intracellular proteins face an enormous
challenge in that they have to cross the plasma membrane. The hydrophobic nature of the
plasma membrane renders it impenetrable to proteins, nucleic acids and hydrophobic
peptides that lack specific membrane receptors or transport proteins. Nevertheless, highly
cationic, naturally occurring peptide sequences of low molecular weight that contains
basic amino acids or proline-rich and completely synthetic molecules, such as model
amphipathic peptides, are able to cross the lipid bilayer [155]. These cell-penetrating
peptides (CPPs) or protein transduction domains (PTDs) efficiently translocate through
Chapter - I
the cell membrane without the need for a receptor, often delivering a hydrophilic cargo
(peptide or protein) into cells. This ability of these domains, together with their low
toxicity, makes them promising potential delivery vectors [156,157]. Their penetrating
capacity depends on the peptide composition and length. CPPs can be classified into two
distinct classes. One class contains predominantly arginine residues (e.g. Antp and Tat),
whereas the other contains predominantly lysine residues [158,159]. CPPs have shown
great potential in a range of therapeutic applications, both in vitro and in vivo. This is
partly owing to the ease with which they can be coupled to nucleic acids, proteins and
small-molecule drugs.
Synthetic peptides are essential tools in various areas of biomedical research, as well as
for the development of diagnostics and pharmaceuticals. Over the past decade, the need
for large numbers of synthetic peptides has lead to the development of synthetic methods
able to generate thousands of individual peptides or peptide analogs for large screening
The peptides or peptide like molecule can serve in principle as building blocks for new
vaccines, diagnostics and drugs. Compared to large recombinant proteins, peptide drugs
offer advantages: they can be made far more specific than biological molecules by
precisely engineered properties due to the absence of constraints imposed by biological
production systems. On top of that peptide drugs will be more robust and, most of all, not
immunogenic. Peptides drugs have, when compared to small molecules, various
advantages: high specificity, often high activity, no accumulations in organs, low toxicity
and no immunogenicity.
Chapter - I
Numerous linear bioactive peptides derived from large proteins, with potential as peptide
drugs, have been around for a long time [160,161,162]. Recently a special subclass, linear
peptide drug candidates derived from antibodies, have been added [163,164,165]. On top
of that multiple peptidelike molecules that mimick complex bioactive sites of large
proteins molecules, have been described recently [166,167,168]. Some of these potential
peptide drugs are in advanced development, while one of them Enfuvirtide [169], a HIV
fusion inhibitor peptide, that represents a complex binding site of gp41 of HIV, is on the
This issue illustrates that peptide drugs are emerging and will form a novel and growing
class of pharmaceutical molecules with exiting new potential, not met by classical drugs
based on small molecules or recombinant proteins [170].
The role of proteins as physiologically active components in the diet is being increasingly
acknowledged. Many of the proteins that occur naturally in raw food materials exert their
physiological action either directly or upon enzymatic hydrolysis in vitro or in vivo. In
recent years it has been recognized that dietary proteins provide a rich source of
biologically active peptides. Such peptides are inactive within the sequence of the parent
protein and can be released in three ways: (a) through hydrolysis by digestive enzymes,
(b) through hydrolysis by proteolytic microorganisms and (c) through the action of
proteolytic enzymes derived from microorganisms or plants. It is now well established
that physiologically active peptides are produced from several food proteins during
gastrointestinal digestion and fermentation of food materials with lactic acid bacteria
[171,172]. Bioactive peptides have been defined as specific protein fragments that have a
positive impact on body functions or conditions and may ultimately influence health
Chapter - I
[173]. Upon oral administration, bioactive peptides, may affect the major body systems
namely, the cardiovascular, digestive, immune and nervous systems depending on their
amino acid sequence. For this reason, the potential of distinct dietary peptide sequences
to promote human health by reducing the risk of chronic diseases or boosting natural
immune protection has aroused a lot of scientific interest over the past few years. These
beneficial health effects may be attributed to numerous known peptide sequences
exhibiting, e.g., antimicrobial, antioxidative, antithrombotic, antihypertensive and
immunomodulatory activities [174]. The activity is based on their inherent amino acid
composition and sequence. The size of active sequences may vary from two to twenty
amino acid residues, and many peptides are known to reveal multifunctional properties
[175]. Today, milk proteins are considered the most important source of bioactive
peptides and an increasing number of bioactive peptides have been identified in milk
protein hydrolysates and fermented dairy products [176,177,178].
1.2.1. Peptides and biological activity
Biologically active peptides range in size from small molecules containing only two or
three amino acids to large molecules containing several tens of amino acids. Among
them, neuropeptides, hypothalamic hormones [179], proteohormones of the pituitary,
immunological significance, peptide vaccines, plasma kinins, atrial nutriuretic peptides,
peptide antibiotics, peptide toxins, peptide insecticides and herbicides are some of the
important classes.
Chapter - I Peptide hormones
Among the plethora of peptide hormones, peptides which are already in clinical use and a
few others described in this section. Among the peptides present in the brain, thyrotropin
releasing hormone (TRH) 1 was the first to be isolated from 1, 00,000 pig hypothalami.
This is the molecule by which the hypothalamus through the pituitary regulates the
functions of the thyroid gland. Later, the first isolation of 800 mg of luteinizing hormone
releasing hormone (LHRH or gona-doliberin, pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-ProGly-NH2) from ventral hypothalami of 1,65,000 pigs was achieved by 12 successive
purification steps by Andrew V Schally and Roger Guillemin, who were awarded the
Nobel Prize for Medicine and Physiology in 1977. LHRH acts on the pituitary to promote
rapid release of LH and follicle-stimulating hormone (FSH), which in turn regulates
ovulation and spermatogenesis. Among several of its clinical applications, LHRH and its
analogs, find use as a nonsteroidal male and female contraceptive or as fertility agents.
Somatostatin, the tetra-decapeptide hormone released by the hypothalamus, plays an
important physiological role as an inhibitor for the release of several hormones
(glucogon, growth hormone, insulin, and gastrin). The octapeptide angiotension II (AspArg-Val-Tyr-Ile-His-Pro-Phe) causes increase in blood pressure. A melanocyte
stimulating hormone (a-MSH) also called as a-melanotropin (Ac-Ser-Tyr-Ser-Met-GluHis-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2), is secreted in the anterior lobe of the pituitary
gland. It exerts its activity at peripheral receptors, which are primarily responsible for its
pigmentation properties. The other hormone secreted in the anterior lobe is a 39 residue
peptide corticotrophin or adrenocorticotropin hormone (ACTH). This finds use in
medicine for the treatment of hypophyseal insufficiencies and inflammatory processes.
Chapter - I
The gastrin family of peptides plays a significant role in gastrointestinal functions
including secretion, motility and absorption. Cholecystokinin is an important hormone,
which belongs to this family mediating digestive functions and feeding behavior. In the
course of the isolation of oxytocin, the second hormone of the pituitary, vasopressin, a
peptide with pressor and antidiuretic effects, was also isolated. The deficiency of
vasopressin causes diabetes insipidus [180]. Persons affected by it have excessive
excretion of water, which is controlled by applying a solution of the peptide in the form
of nasal spray. The vital regulation of the level of Ca2+ ions in the blood is controlled by
parathyroid hormones. These hormones raise the level of calcium concentration in blood
by mobilizing calcium uptake from the bones. On the other hand, the thyroid hormone,
calcitonin directs the flow of calcium ions in the opposite direction. Thus a constant level
of calcium ions is maintained in the blood.
[Primary structure of TRH (pGlu-His-Pro-NH2)] Neuropeptides
There are about 50 neuropeptides whose molecular structures are known; many of
them exist within closely related families of molecules. Several endogenous opioid
pep-tides [endorphins, enkephalins, dynorphins, dermorphins, deltorphins, dermorphin
gene associated peptide (DGAD)] possessing morphinomimetic properties are very well
Chapter - I
studied. Enkephalins (meaning isolated from the brain), two pentapeptides (Tyr-Gly-GlyPhe-Met/Leu) are found in varying amounts in almost all regions of nervous system. A
large number of their analogs have been synthesized to find out their role in pain
transmission, since they act as transmitters of the paininhibitory neurons on the spinal
cord. These brain morphins have been well studied which is known to allow analgesia to
be separated from the development of addiction and dependence.
Gramicidin S
(Active against gram-positive but not against gram-negative bacteria) Peptide leukotrienes:
The peptide leukotrienes cause contraction of the bronchial smooth muscle and probably
play an important role as mediators in allergic reactions (eg. asthma) and inflammations. Peptide antibiotics:
The peptidic nature of penicillins has long been recognised. Penicillins and
cephalosporins inhibit final stages of the enzymatic construction of the bacterial
peptidoglycan cell wall component, a network of peptides and polysaccharides. The
Chapter - I
depsipeptide, valinomycin has been found to be active against a number of bacteria,
yeasts and fungi. Peptides of immunological significance:
Immune responses to synthetic peptides constitute another imporant field of study.
Serums raised against synthetic peptides are highly specific reagents for the
corresponding native proteins from which the peptide amino acid sequence was derived.
These antipeptide serums are powerful reagents for the detection and characterization of
Human vasopressin (antidiuretic harmone)
Human Oxytocin (Uterine contraction)
Two hormones 3 & 4 of the posterior pituitary gland are identical in all but two residues
exhibit very different biological activity Poisonous peptides:
The most important component of snake venoms are polypeptide neurotoxins such as
cobrotoxin, which produce lethal flaccid paralysis through a neuromuscle block. The
peptides of amanita mushrooms such as antamanides are among the best known peptide
poisons. The smallest dose of 50 mg can kill a 20 gm mouse within a few hours. (2.5 mg
Chapter - I
/kg of body weight). A search for inhibitors or antidotes through antagonists can be
predicated. Others:
Actinomycin belongs to a class of chromopeptides and is characterized by its cytostatic
growth inhibition in tumors and for antibacterial action. The cyclic despipeptides,
destruxins act as insecticides whereas bialaphos (phosphinotricylalanyl alanine) has a
strong herbicidal action. Peptides can be either sweet or bitter. The bitter peptide ArgGly-Pro-Pro-Ile-Val, isolated from casein hydrolysates and delicious tasting peptides
from fish proteins has gained practical importance in the food industry. Nisin, produced
by Streptococcus lactis is used as a food preservative. The antrial natriuretic peptides
have diuretic properties. The phosphopeptide, (S)-alanyl-(R)-1-aminoethyl-phosphoric
acid (alafosfalin) is effective against both gram positive and gram negative
microorganisms. Albomycins are nucleopeptides which possess iron-complexing
properties. Several muramyl peptides (glycopeptides) have the possibility of being used
as adjuvants in combination with vaccines or with antibiotics.
1.2.2. Examples of peptides and their bioactivity Somatostatin
Somatostatin analogue infusion improved memory for patients with AD, perhaps through
modulation of the insulin content Neuropeptide Y (NPY)
It may be involved in aluminium metabolism in animal models, and aluminium
accumulation has been associated as a risk factor for AD, mainly in combination with
Chapter - I Galanin
Galanin levels increase with the duration of AD. Galanin inhibits cholinergic
transmission and long term potentiation in hippocampus. Galanin’s excitatory action on
cholinergic neurons may play a compensatory role by augmenting the release of
acetylcholine from remaining cholinergic basal forebrain neurons. Insulin like growth factor I (IGF I)
IGF I protects in vitro primary neurons from cytotoxic mechanism of the London type
Abeta PP mutant. Interleukin-6 and interleukin-11
Both interleukins attenuate cytotoxicity of the London type Abeta PP mutant. Apoptosis-antagonizing transcription factor (AATF)
AATF protects neurons against Abeta-induced apoptosis in PC 12 cells. SAL (SALLRSIPA)
SAL is active fragment of ADNF and prevents neuronal cell death produced by electrical
blockade, N-methyl-Daspartate, and Abeta. Activity -dependent neuroprotective protein (ADNP)
The protein implicated in maintenance of cell survival through modulation of p53
expression. The ADNP was identified as a molecule that may mediate protection offered
by lipophilic analogues against ischemia cell death. Bcl-w
Bcl-w is a member of the Bcl-2 anti-apoptic protein family that promotes cell survival,
significantly protects neurons against stauroporine and Abeta induced apoptosis.
Chapter - I Gly-Pro-Arg
This tripeptide effectively protects and rescues cell death induced by Abeta. RER peptides
Peptides containing the palindromic tripeptide RER sequence (Arg-Glu-Arg), present in
the amyloid precursor protein, protects against memory loss cause by Abeta and acts as a
cognitive enhancer. Autocamtide-related inhibitory peptides (AIP)
These peptides inhibit Ca/Calmodulin dependent protein kinase II, inhibit Abeta triggered
activation of caspase 2 and 3, decrease tau phosphorylation and protect neuron against
Abeta toxicity. Substance P
This short peptide interacts with cholinergic ascending system of the nucleus basalis
Meynert, resulting enhancement effects. Patients with AD show a marked loss of
cholinergic neurons and diminished brain substance P expression.
Synthetic peptides are essential tools in various areas of biomedical research, as well as
for the development of diagnostics and pharmaceuticals. Over the past decade, the need
for large number of synthetic peptides has lead to the development of synthetic methods
able to generate thousands of individual peptides or peptide analogs for large screening
programs [181].
There are multiple examples in nature, where peptides are used as ligands to influence the
function of specifically binding target proteins. Also, cellular regulatory mechanisms are
dependent on numerous inhibitory proteins that function through allosterism or inhibition
of protein interactions.
Chapter - I
Currently, proteins and peptides experimentally selected for high affinity intracellular
interactions with pre-determined target structures are emerging as important molecules,
which could serve to extend conventional druggability. In a few model systems, peptides
have already been used to manipulate crucial regulatory networks in cancer cells
[182,183,184,185]. They can target specific intracellular proteins required by cancer cells
for proliferation and invasion. Additional essential signalling components in cancer cells
are being discovered and it is conceivable that individual peptides can be derived to
inhibit their function in a targeted fashion. These peptides can be used for mono therapy
or in combination with conventional chemotherapeutic agents. Since multiple pathways
are dysfunctional in different cancers, and cancer cells accumulate oncogenic mutations
as they progress, the greatest and most durable benefit will likely be achieved by
combining therapeutic agents, which address different hallmarks of cancer. This concept,
also called ‘multi-focal signal modulation therapy’ (MSMT), is promising, since
combinations of signal modulators have already achieved dramatic suppression of tumour
growth [186,187,188]
This is exemplified by the design of a peptide binding to TRAF6 (tumour necrosis factor
(TNF) receptor associated factor 6), which was derived from the sequence of two natural
binders. TRAF6 participates in signal transduction mediated by TNF receptors as well as
the IL-1 receptor family and plays a role in immunity and bone homeostasis. This peptide
inhibits TRAF6 signalling and osteoclast differentiation and has the potential as a
therapeutic modulator for the treatment of osteoporosis or cancer-induced bone lesions.
The use of peptides as drugs is limited by a number of biological properties, which have
to be altered and manipulated in order to enhance efficacy. These include their metabolic
Chapter - I
instability, the inability to cross cell membranes and their potential immunogenicity. In
the past years, considerable progress has been made to convert peptides into
therapeutically useful molecules. This includes the increase in stability by chemical
modifications. Peptides assembled partly or totally from D-amino acids are more stable
and less susceptible to proteolytic degradation than peptides made from natural L-amino
acids. D-peptides are obtained by synthesising D-amino acids in the reverse order from
those in the parent peptide and replacing L-amino acids. They usually closely resemble
the structure of the L-variants or even show increased affinity [189,190,191]. Short
peptides (<40 mer) can be chemically synthesised on a small scale. Chemical
modifications of the peptides can be also incorporated to increase the binding efficacy.
This has been achieved with non naturally occurring or phosphorylated amino acids or by
protection of the N- and C-terminus. This improved the systemic and intracellular
stability of the recombinant peptides. However, synthesis of longer and highly structured
peptides is laborious and expensive. E. coli and insect systems are now available, which
allow peptides and proteins to be obtained in a recombinant setting. With such systems it
is even possible to incorporate non-natural amino acids into proteins or to modify coded
ones by post-translational modifications [192,193].
A peptide molecule exists in either a compact folded state or a variable and open
unfolded state. One way of shifting the equilibrium toward the folded state is by inserting
peptides with both ends in a platform or a so called scaffold protein or by attaching them
on one side to a support protein (like SUMO-1). This not only increases stability but also
constrains the conformation, improving binding affinities by decreasing their flexibility
in solution (entropy) before binding [194]. For most screening methods mentioned above,
Chapter - I
peptides are usually presented in a constrained setting. Critical factors in the development
of effective scaffolds that can be used in cancer therapy include absence of regions prone
to aggregation or susceptible to proteolysis. The scaffold should also offer low
immunogenicity, high affinity and specificity, solubility and stability to the peptide.
Meanwhile multiple scaffolds have been described, each displaying their own advantages
and applicabilities.
One of the most popular scaffolds for peptide library display is the bacterial thioredoxin
A protein (Trx). This protein is characterised by a stable structure and can be easily
purified in large quantities from E. coli extracts. Efficient purification is an important
parameter for determination of aptamer structure as well as for protein transduction
experiments. It might become the basis for cost effective production of potential
therapeutics. Numerous peptide aptamers integrated into this scaffold have been
identified, which bind to various target proteins [195,196,197,198]. It was even possible
to show that the binding domain of a natural interaction partner can be displayed in a
proper interacting conformation by the Trx scaffold [199]. Other proteins that are suitable
for the presentation of peptides, a catalytically inactive derivative of Staphylococcus
nuclease, and the cellular transcription factor SP1 have been described [200,201].
The recent reports suggest that cell-penetrating peptides might be able to transport
macromolecules across the blood brain barrier [202].
The peptides or peptide like molecule can serve in principle as building blocks for new
vaccines, diagnostics and drugs. Compared to large recombinant proteins, peptide drugs
offer advantages: they can be made for more specific than biological molecules by
precisely engineered properties due to the absence of constraints imposed by biological
Chapter - I
production systems. On top of that peptide drugs will be more robust and most of all not
Peptides drugs have, when compared to small molecules, various advantages: high
specificity, often high activity, no accumulations in organs, low toxicity and no
Various approaches have also been used to rigidly an active structure by cyclization or by
introduction of constraints into regular peptide molecules in order to amplify
conformational and functional features beneficial to enzymatic stability and biological
activity. Diverse peptidomimetics have found applications in immunology [203] receptor
binding interactions [204] and in the design of enzyme inhibitors [205]. The development
of conceptual approaches for synthetic modification of the molecule in order (i) to endow
the analog ideally with only one specific activity (i.e to change the activity profile of the
peptide so that only the desired biological property is expressed, whether agonistic (or
antagonistic) and (ii) to increase the resistance of the peptide to enzymatic inactivation
and to alter tissue distribution, with the hope of prolonging the duration of hormonal
action [206].
The neuroprotective potential of different peptides has become a matter of intensive
investigation in many animal models. Many in vitro studies reveal that peptides protect
neurons against apoptosis occurring naturally during CNS development and apoptosis
induced by a series of neurotoxins, prion protein, Abeta, HIV envelope glycoprotein
(gp120), potassium ion deficit and high glutamate concentrations [207].
Chapter - I
The development and successful application of soluble peptide combinatorial libraries
have demonstrated the power of combinatorial approaches for basic research and for the
identification of highly active compounds having therapeutic potential [208].
Proteins and peptides are fundamental in life with an extensive array of functions born of
the multitude of possible structures. Consisting of common fragments the primary,
secondary and tertiary structures formed through intra- and inter-molecular interactions
provide innumerable variations of structure and resultant activity in biological systems.
However, the serial nature of synthetic peptide synthesis leads to the perpetual problem
of long synthetic routes when investigating residue effects through analogue synthesis.
While the ability to modify individual residues of peptides and complex molecules
selectively and reliably leads to a number of exciting possibilities in chemical biology
and synthetic strategies. Currently, the majority of methods applied to the modification of
biological systems are based on biochemical techniques [209], however, examples of
modifications achieved through small molecule (chemical) reactivity is increasing [210].
Peptides have multiple biological actions in the brain, and are potentially valuable as
neuropharmaceuticals in the treatment of various disorders of the CNS. Possible roles of
peptides in the CNS include; 1) involvement in neurotransmission and neuromodulation;
2) regulation of the neuroendocrine axis; 3) regulation of cerebral blood flow; 4)
regulation of (cerebro spinal fluid) CSF secretion; 5) mediation of the integrity of the
BBB; 6) modulation of the BBB permeability to nutrients; 7) regulation of water and
electrolyte contents of the brain; 8) regulation of the expression of specific proteins at the
BBB [211].
Chapter - I
Thus, peptide drugs may be useful to treat or to diagnose brain disease. However,
delivery of peptide drugs to the brain is an essential prerequisite for therapeutic
effectiveness, since distribution of peptides and proteins to the brain is generally very low
because of the blood-brain barrier (BBB) that prevents many molecules from crossing
into the brain.
1.3. Introduction to heterocyclic compounds
1.3.1. Introduction to benzimidazole derivatives
One of the goals of medicinal chemistry research and drug discovery is to provide a
rational basis for the design of new medicinal agents. Organic compounds and their
reactions have been utilized by people since antiquity. When leaves or tree bark or plant
roots are mixed with water to make a medicinal potion, a complex mixture of organic
products is actually extracted for its biologically active components. In 1960s, a broad
spectrum group of drugs, known as benzimidazoles, were discovered with a big-gang
having specific activity. Due to the increasing demand for bioactive molecules, organic
chemists are increasingly required to synthesize new compounds of biological interest.
There has been an unlimited expansion of molecular diversity in synthetic organic
compounds by the application of combinatorial methodology. The benzimidazole nucleus
is an important pharmacophore in drug discovery [212] and it is a fused aromatic
imidazole ring where a benzene ring is fused to 4 and 5 positions of an imidazole ring.
Chapter - I
Benzimidazoles are very useful intermediates for the development of molecules of
pharmaceutical or biological interest. Substituted benzimidazole derivatives have found
applications as in diverse therapeutic agents including antiulcer, antihelmintic,
antihypertensive, anticoagulant, antiallergic, analgesic, antiinflammatory, antipyretic,
antianxiolytic. Because of their significant medicinal importance, the synthesis of
substituted benzimidazoles has become a focus of synthetic organic chemistry.
Benzimidazoles are listed with various effects on human body and are used to treat
multiple system disorders. Benzimidazole derivatives as anticancer agents
Cancer is most common cause of death next only to heart disease. Accounting for 22.3%.
The cancer death rate per 100,000 populations in 1930 was 143; in 1950, it was 157; and
in 1986, it was 171. Lung cancer was the major cause for this increase, and in 1986, the
deaths from lung cancer were approaching 50% of all reported deaths from cancer. Most
multicellular organisms can be afflicted by cancer and cancerous lesions have been found
in dinosaur bones. Only in the twentieth century, however, has there been much concern
over the disease. Progress in the cure of the former major causes of death has inevitably
led to a risen in the incidence of cancer.
The outlook for survival rates from cancer shows improvement. Those who are alive 5
years after diagnosis were 1 in 5 in the 1930s, 1 in 4 in the 1940s, and 1 in 3 in the 1960s.
More recently, 4 in 10 patients survive 5 years. In terms of normal life expectancy, 50%
of cancer patients are now alive after 5 years. Poor Americans, regardless of race, have a
5-year survival rate that is 10 to 15% lower than the average [213].
Chapter - I
Tapas Mukhopadhyay et. al., reported the Mebendazole, a derivative of benzimidazole,
induces a dose and time-dependent apoptotic response in human lung cancer cell lines.
Mebendazole arrested cells at the G2-M phase before the onset of apoptosis, as detected
by using fluorescence-activated cell sorter analysis [214]. Xiaofen Huang et. al., reported
the antitumor (melanoma, non-small-cell lung) activity of Pyrrolo[1,2-a]benzimidazoles
[215]. Laura Garuti et. al., reported the antiproliferative activity of some benzimidazole4, 7-dione derivatives, compound 6 possess the best antiproliferative effect. Such activity
is strong against SUPT1 cells, although lower than that of the MMC. Moreover,
compound 6 is more active than MMC on human lymphoblastic leukemia, whereas high
antiproliferative activity of 6 on leukemia and lymphoma cells [216].
The DNA minor groove binders, Hoechst 33258 and Hoechst 33342, have been reported
to protect against radiation-induced DNA-strand breakage, but their mutagenicity and
cytotoxicity limit their use as protectors of normal tissue during radiotherapy and as
biological radioprotectors during accidental radiation exposure [217,218]. Dyes Hoechst
33258 and Hoechst 33342 are frequently used in cytometry to stain chromosomal
materials in situ because these two compounds become highly fluorescent upon bonding
to DNA [219].
Edmunds Lukevics et. al., reported the antitumour activity of trimethylsilylpropyl
substituted benzimidazoles, N-trimethylsilylpropylbenzimidazole (8) exhibits high
cytotoxic activity [220]. John Mann et. al., reported the antitumor activity of symmetric
bisbenzimidazole-based DNA minor groove-binding agents, the compound 2,2-bis-[4¢(3¢¢-dimethylamino-1¢¢-propyloxy)phenyl]-5,5-bi-1H-benzimidazole possesses promising
Chapter - I
in vitro antitumor growth inhibitory properties, with the concentration required for 50%
inhibition being around 200-300 nM [221].
Seref Demirayak et. al., reported the anticancer activity of 1-methylene-2,3-diaryl-1,2dihydropyrazino[1,2-a]benzimidazole
a]benzimidazole derivatives [222]. Ahmed Kamal et. al., reported the DNA-binding
affinity and in vitro anticancer activity of C8-linked pyrrolo[2,1-c] [1,4] benzodiazepine–
benzimidazole conjugates [223]. El-Sayed A. M. reported the antineoplastic activity of
cycloalkyl pyrido[1, 2-a] benzimidazoles, the p-fluorophenylamino derivative 10 was the
most active candidate against K-562, Molt-4 and RPMI-8226 leukaemic cell lines [224].
Antonio Da Settimoa et. al., reported the DNA binding and in vitro antiproliferative
activity of purinoquinazoline, pyridopyrimidopurine and pyridopyrimidobenzimidazole
derivatives [225].
Shu-Ting Huang et. al., reported the anticancer evaluation of bis benzimidazoles [226].
Chandra Kumar et. al., reported the inhibition of angiogenesis and tumor growth by
benzimidazole derivatives [227]. Kristina Starcevic et. al., reported the antitumor activity
of 2-substituted-5-amidino-benzimidazoles. The most pronounced antiproliferative
activity was shown with compounds having imidazolinylamidino-substituent [228].
Interestingly another promising group of antitumor compounds is represented by
benzimidazo[t,2-c]quinazolines and thiazolo[3,4-a]benzimidazoles [229,230].
Chronic lymphocytic leukemia is a cancer of the white blood cells (lymphocytes).
Treanda is indicated for the treatment of patients with chronic lymphocytic leukemia.
Chapter - I
Hoechst 33258
O 6
Hoechst 33342
Benzimidazole derivatives with potent anticancer activity and Hoechst 33258 and
Hoechst 33342, are used to protect against radiation-induced DNA-strand breakage
The more spectacular advances in medicinal chemistry have been made when
heterocyclic compounds played an important role in regulating biological activities.
Among various heterocycles, sulfur-nitrogen heterocycles have maintained the interest of
reaserchers through decades of historical development of organic synthesis. The grounds
of this interest were their biological activities and unique structures that led several
applications in different areas of pharmaceutical and agrochemical research or, more
recently in material sciences [231].
Chapter - I
1.3.2. Introduction to morpholine arecholine derivatives
Arecoline is a natural organic compound which is an alkaloid found in betel nuts from the
betel palm (Areca catechu) It is an oily liquid that is soluble in water, alcohols, and ether
In many Asian cultures, the betel nut is chewed to obtain a stimulating effect. Arecoline
is the primary active ingredient responsible for the central nervous system affects which
are roughly comparable to those of nicotine, which has a similar chemical structure.
Arecoline is known to be an agonist of muscarinic M1, M2 and M3 receptors [233], which
is believed to be the primary cause of its parasympathetic effects (such as pupillary
constriction, bronchial constriction, etc.). Arecoline has also been used medicinally as an
antihelmintic [234] (a drug against parasitic worms).
Pedersen et al [235] have investigated muscarinic receptor affinity as well as estimated
relative efficacy and subtype selectivity of bicyclic arecoline bioisosteres 13(a-d) using
rat brain membranes and a number of tritiated muscarinic receptor ligands.
a: CH3
b: C2H5
c: -CH2-CH=CH2
d: -CH2CN
Chapter - I
The effects at the five cloned human muscarinic receptor subtypes of a selected series of
chiral analogues, with established absolute stereochemistry, were studied using receptor
selection and implication technology. The potency, relative efficacy, and receptor
subtype selectivity of these compounds were related to the structure of the O-substituents
and the position and stereo chemical orientation of the piperidine ring methyl
A series of derivatives of arecoline were synthesized in an effort to develop M1
muscarinic agonists [236]. The arecoline derivatives stimulated phosphoinositide
turnover through muscarinic receptors in the rat hippocampus. Molecular mechanics
calculations indicate that the anti form of the 1,2,5-thiadiazole derivatives of arecoline
may be active at M1 receptors.
In addition, a series of arecoline derivatives were synthesized and tested for muscarinic
activity in receptor binding assays using [3H]-oxotremorine-M (3H-OXO-M) and [3H]pirenzepine (3H-pZ) as ligands. Potential muscarinic agonistic or antagonistic properties
of the compounds were determined using binding studies measuring their potencies to
inhibit the binding of 3H-OXO-M and 3H-PZ. Preferential inhibition of 3H-OXO-M
binding was used as an indicator for potential muscarinic agonistic properties; this
potential was confirmed in functional studies on isolated organs. All compounds with
agonistic properties showed 3H-pZ / 3H-OXO-M potency ratios in excess of 20.
Chapter - I
In contrast, for antagonists this ratio was found to be close to unity. Mono-halogenation
resulted in compounds (14a and 14b) with M3 agonistic properties as shown by their
atropine sensitive stimulant properties in the guinea pig ileum, but with very little or no
Ml activity. Some minor in vivo effects were observed for both these compounds, with
the iodinated compound 14b inducing salivation. Compound 14a also showed some
positive mnemonic properties in rats where spatial short-term memory had been
compromised by temporary cholinergic depletion. These data indicate that some M3
agonism may be desired in therapeutic agents aimed at the treatment of the cognitive
deficits of Alzheimer's disease patients.
1.3.3. Introduction to 1,2-benzisoxazoles derivatives
Many reports ascribe interesting biological activities of 1,2-benzisoxazoles and their
derivatives. The chemistry of substituted 1,2-benzisoxazole amides occupies an
extremely important role in the field of pharmaceuticals and in medicinal fields.
Compounds containing amide bond, benzisoxazoles, chromans and fluorine atom
substitution can alter the chemical properties, disposition, and biological activities of
drugs [237]. Many fluorinated compounds, 1,2-benzisoxazole derivatives and various
amides are currently used in the treatment of diseases [238]. These include,
antidepressants, anti-inflammatory agents, antimalarial drugs, antipsychotics, antiviral
agents, steroids, and general anaesthetics. 1,2-benzisoxazole ring containing in the drugs
namely Zonisamide (15) and Risperidone (16) an anticonvulasants [239]. The fluorine
substitution can also have a profound effect on drug disposition, in terms of distribution,
drug clearance, route(s), and extent of drug metabolism [240].
Chapter - I
Benzisoxazoles are currently the most important building blocks in drug discovery, with
a high number of positive hits encountered in biological screens of this heterocycle and
its congeners. The benzisoxazole template forms the molecular backbone, possesses
versatile binding properties with a frequently occurring binding motif, and provides
potent and selective ligands for a range of different biological targets in Medicinal
Chemistry. The benzisoxazole scaffold and its analogues are important pharmacophores
that can be found in biologically active compounds across a number of different
therapeutic areas such as anti-HIV [241], anticancer [242], anti-inflammatory [243],
dopamine and serotonin receptors [244], anticonvulsants [245], acetylcholineesterase
[246] and antimicrobial [247].
1.4. Scope of the present work
The study of heterocycles is an evergreen field in the branch of organic chemistry and
always attracts the attention of scientists working not only in the area of natural products
but also in the synthetic organic and medicinal chemistry. Moreover, many useful drugs
have emerged from the successful investigations carried out in this branch. Besides,
spectacular advances have been made to furtherance the knowledge of relationship
between chemical structure and biological activity. In fact, this tendency is reflected by
the voluminous data available in literature on heterocyclic chemistry.
Chapter - I
There is a large amount of literature dealing with the physiological significance of free
amino acids and their effects on a variety of metabolic and physiological systems. Amino
acids and their metabolic and physiological ramifications are among the most
investigated topics in biomedical science. The synthesis of substituted amino acids has
attracted the attention of chemists due to their biological activities and the interesting
structural properties of their molecules. For example NS5B polymerase inhibitors against
the hepatitis C virus, anti inflammatory bradykinin B1 receptor antagonists, anticancer
matrix metalloproteinase (MMP-12) inhibitors or analgesic endomorphin-1 analogue
tetrapeptides. (S)- β-Phenylalanine has been applied in the synthesis of novel antibiotics.
Some amino acids are reported to have strong antioxidant activity in linoleic acid and
methyl linoleate model systems.
The salient features of the usefulness of conjugation of amino acids with drugs are as
follows. (i) Amino acids are normal dietary constituent and they are non-toxic in
moderate doses as compared to other promoities; (ii) amino acids have healing effect on
gastric toxicity; (iii) being a nutritional substance, the use of amino acids as a derivatizing
group might also permit more specific targeting site for enzymes involved in the terminal
phase of digestion; (iv) many amino acids possess marked anti-inflammatory activity
against carrageenan induced hind paw edema in rats; and (v) by using different types of
amino acids, viz. non-polar, polar, acidic and basic, the drug molecule can be made more
or less polar, or more or less soluble in given solvent
One of the most important problems of modem organic chemistry is the search for highly
effective bioregulators having a broad spectrum of biological action at comparatively low
toxicity. Modification of the structure of heterocyclic compounds by conjugate with
Chapter - I
amino acids and peptides is one of the routes for constructing new classes of biologically
active substances. Introducing an amino acid or peptide into a heterocyclic compound can
increase the hydrophilicity or lipophilicity, decrease the toxicity, and prolong its action.
Furthermore, such modification can change the selectivity of its action. The conjugation
of heterocyclic compounds with peptides leads to potent analgesics, highly active
antimicrobials, enzyme inhibitors, highly specific antigenic determinants, and inhibitors
of melittin's hemolytic activity.
Peptides conjugated drugs have multiple biological actions in the brain, and are
potentially valuable as neuropharmaceuticals in the treatment of various disorders of the
CNS. Possible roles of peptides and its conjugates in the CNS include; 1) involvement in
neurotransmission and neuromodulation; 2) regulation of the neuroendocrine axis; 3)
regulation of cerebral blood flow; 4) regulation of cerebro spinal fluide (CSF) secretion;
5) mediation of the integrity of the Blood brain barrier (BBB); 6) modulation of the BBB
permeability to nutrients; 7) regulation of water and electrolyte contents of the brain; 8)
regulation of the expression of specific proteins at the BBB.
Thus, the successful applications in various fields ensure a limitless scope for the
development of structurally novel amino acid and peptide conjugates of heterocyclic
compounds with a wide range of physico-chemical and biological properties. Hence this
prompted us to synthesize novel amino acid conjugates of 1H-benzimidazoles and 4piperidinyl-1,2-benzisoxazole and peptide conjugates of 3-morpholino Arecoline to
investigate their diverse biological activities such as anti-cancer, anti-convulsant, antiDNA damaging and anti-Alzheimer’s disease.
Chapter - I
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